Improved optical transmission sample holder and analysis, particularly for hemoglobin

ABSTRACT

Among other things, the present invention is related to devices and methods for improving optical analysis of a thin layer of a sample sandwiched between containing between two plates.

CROSS REFERENCE

This application is a National Stage entry (§ 371) application ofInternational Application No. PCT/US18/65874, filed on Dec. 14, 2018,which claims the benefit of U.S. Provisional Patent Application No.62/598,899, filed on Dec. 14, 2017, the contents of which are reliedupon and incorporated herein by reference in their entirety.

The entire disclosure of any publication or patent document mentionedherein is entirely incorporated by reference.

FIELD

The present disclosure is related to devices and methods for improvingoptical transmission analysis of a thin layer of a sample sandwichedbetween containing between two plates.

BACKGROUND

An optical absorption by a thin layer of a sample is one of the methodsto assay a biological and chemical sample. One way to measure an opticalabsorption is to measure the intensity of the incident light and thetransmitted light that directly goes in and out of a sample,respectively.

However, in many practical situations, it can be difficult to directlymeasure these light intensities, because of various reasons. One reasonis that a thin layer sample often needs a sample holder for ameasurement and the transmitted light being measured is the light thatgoes through both the sample and the sample holder. Hence, there is aneed for a method that can separate the light absorption by the sampleholder from that by the sample.

Another reason is that the incident light and transmitted light are onthe opposite side of a sample, it is difficult to use a single detectorto both light. Hence, there is a need for using a single photodetectorfor an absorption measurement.

In prior approaches of optical transmission measurement of a thinsample, a sample holder that comprises two plates has been used tosandwich a sample into a thin layer between the two plates, and thelight transmission through an air bubble inside the sample thin layer(which can occur under certain conditions) was used as a referencesignal to separate the light absorption by the sample holder from thatby the sample. This approach also allow an optical absorptionmeasurement with a single photodetector. In the method, it assumes that(i) light transmission through the air bubble area is the same as thatthrough a zero thickness sample, and (ii) light absorption by the sampleholder is the same in the air bubble area (where the reference signal ismeasured) and in the sample area (where the sample single is measured).However, in reality, both assumptions can be wrong. An air bubble can begenerated significantly away from the location of the sample signal, sothat there is a significant difference in sample holder absorptionsbetween two locations. The air bobble can be too small, so that lightwill be significantly scattered and the reference signal issignificantly different from a sample having zero thickness.Furthermore, the air bubble generation is random in both occurrences(can or cannot occur) and the location (e.g., random locations).

Accordingly, an object of the present invention to provide the devicesand methods to generate the reference light, simplify the opticaltransmission measurement, and simplify a sample handling. The presentinvention can overcome or reduce the disadvantages of the prior devicesor systems.

BRIEF SUMMARY

Among other things, the present invention is related to devices andmethods for improving optical analysis of a thin layer of a samplesandwiched between containing between two plates, particularly, forgenerating a reference signal that can improve the optical analysis, andfor an application of assaying hemoglobin.

A property (e.g. a biological or chemical property) of a sample can bedetermined by the optical density (i.e. OD) of the sample by the ratioof the intensity of the transmitted light through a thin sample layer tothe incident light (i.e. the Beer-Lambert's Law). However, a thin layersample often needs a sample holder for a measurement, and the lightbeing measured also goes through the sample holder. There is a need toseparate the optical transmission signal and optical absorption (e.g.optical density) of a sample from the total transmitted light, whichinclude the light transmission through the sample and through the sampleholder.

One objective of the present invention provides the devices and methodsof certain embodiments of a sample holder and the use of that improvesthe optical transmission measurements.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

A skilled artisan will understand that the drawings, described below,are for illustration purposes only. In some Figures, the drawings are inscale. In the figures that present experimental data points, the linesthat connect the data points are for guiding a view of the data only andhave no other means. For clarity purposes, some elements are enlargedwhen illustrated in the Figures. It should be noted that the Figures donot intend to show the elements in strict proportion. The dimensions ofthe elements should be delineated from the descriptions herein providedand incorporated by reference. The drawings are not intended to limitthe scope of the present invention in any way.

FIG. 1A illustrates a cross-section view of one embodiment of a sampleholder, termed OAC (e.g. optical analysis card), for analyzing ananalyte in a sample (e.g. hemoglobin in a blood sample) by opticaltransmission using light, comprising: a first plate, a second plate, anda light guiding spacer (LGS); wherein the LGS has a pillar shape, issandwiched between the two plates with each end of the pillar in directcontact to one of the plates forming a LGS-plate contact area, and isconfigured to allow the light transmits from the first plate, throughthe LGS, to the second plate without going through a sample.

FIG. 1B illustrates a cross-section view of one embodiment of a sampleholder, termed OAC (e.g. optical analysis card), for analyzing ananalyte in a sample (e.g. hemoglobin in a blood sample) by opticaltransmission using light, comprising: a first plate, a second plate, anda light guiding spacer (LGS), wherein the two plates are movablerelative to each other and the pillar has flat top.

FIG. 1C is an illustration of a CROF (Compressed Regulated Open Flow)embodiment. Panel (a) illustrates a first plate and a second platewherein the first plate has spacers. Panel (b) illustrates depositing asample on the first plate (shown), or the second plate (not shown), orboth (not shown) at an open configuration. Panel (c) illustrates (i)using the two plates to spread the sample (the sample flow between theplates) and reduce the sample thickness, and (ii) using the spacers andthe plate to regulate the sample thickness at the closed configuration.The inner surface of each plate may have one or a plurality of bindingsites and or storage sites (not shown).

FIG. 2A illustrates a cross-section view of one embodiment of a sampleholder with a first plate, second plate and a LGS, a sample in theholder, locations of a sampling region and a reference region, andincident light and transmitted light in the sample region and thereference region respectively

FIG. 2B illustrates a perspective view of one embodiment of a sampleholder with a first plate, second plate and a LGS, a sample in theholder, and locations of a sampling region and a reference regions.

FIG. 3 illustrates a top view of one embodiment of a sample holder witha first plate, second plate and a LGS, location of a LGS, a samplingregion, a reference region, and an exemplary location of the edge of thereference region and the sample region. Note that the edges are selectedduring an imaging processing.

FIG. 4 illustrates the molar extinction coefficient of Oxygenatedhemoglobin [HbO₂] and deoxygenated hemoglobin [Hb] at wavelength 200 nmto 1000 nm.

FIG. 5 illustrates an optical setup for measuring hemoglobin in QMAXcard.

FIG. 6 illustrates an exemplary hemoglobin measurement in a QMAX cardtaken by an iPhone.

FIG. 7 illustrates an exemplary QMAX hemoglobin measurement comparedwith the gold standard (Abbott Emerald Hemocytometer).

FIG. 8 illustrates an exemplary hemoglobin measurement in a QMAX cardtaken by iPhone.

FIG. 9 illustrates an exemplary method for selecting sampling regionsand reference regions.

DETAILED DESCRIPTION

The following detailed description illustrates certain embodiments ofthe invention by way of example and not by way of limitation. If any,the section headings and any subtitles used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described in any way. The contents under a sectionheading and/or subtitle are not limited to the section heading and/orsubtitle, but apply to the entire description of the present invention.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The terms “light guiding spacer” or “LGS’ can refer to a pillar that,during an optical transmission measurement of a sample, has one end ofthe pillar in direct contact to a first plate and the other end of thepillar in direct contact of a second plate, In certain embodiments, thefirst plate and the second plate sandwich the sample between the twoplates. In certain embodiments, the optical index and the size of thepillar are predetermined and known. In certain embodiments, the LGS ismade of the same material as one or both of the plate. In certainembodiments, the LGS is bond, mold, imprinted, or other ways toconnected to one or both plate.

The term “no significant amount of sample” can refer to an amount ofsample that is insignificant to an optical transmission measurement ofthe sample when the measurement is performed in an area that has the twoplates and the sample.

The term “LGS-Plate contact areas” can refer to the area in each end ofthe LGS (which has a pillar shape) that is in direct contact to one ofthe plates. In certain embodiments, the LGS and one plate is made in onepiece of a material, then the LGS-Plate contact area for the end of theLGS connected to the plate is the cross-section of the LGS. In certainembodiments, the LDG and both plates are made of a single piece ofmaterial, then the LGS-Plate contact area for both end of the LGS is thecross-section of the LGS.

The terms “lateral cross-section of the LGS” can refer to that across-section of a LGS that is parallel with the plates when the LGS issandwiched between the two plates. The terms of “the LGS-contact area ora lateral cross-section of the LGS are larger than the wavelength of thelight” can refer to that the LGS-contact area or a lateral cross-sectionof the LGS are larger than the wavelength of the light is larger thanthe area of disk that has a diameter equal to the wavelength of thelight.

The terms “OTSA” means optical transmission sample analysis, thatmeasures the optical density of a thin sample layer by opticaltransmission.

The term “a SR region” or “a pair of SR region”, which areinterchangeable, can refer to one sampling region and one correspondingreference region, where an OD of a thin sample layer is determined bytaking a ratio of the intensities of the light transmitted through thesample region and through the reference region.

The term “reference region” of an OAC device can refer to the region ofthe device where light of a wavelength and a polarization goes throughthe first plate, the light-guiding spacer, and the second plate, whereinthe light guiding spacer is a direct contact of the first and secondplates. The term “reference region” of an OAC device can refer to theregion of the device where a light guiding spacer is sandwich betweenthe two plates and has a direct contact respectively to each plate,wherein, in the reference region, a probing light transmits through, insequence, the first plate, the light-guiding spacer, and the secondplate, without going through the sample.

The term “sampling region” of an OAC device can refer to the region ofthe device where the light of the sample wavelength and thepolarization, that goes through the reference region, goes through thefirst plate, a sample between the two plates, and the second platewithout going through the light guiding spacer.

The term “sampling region” of an OAC device can refer to the region ofthe device where the sample is between the two plates without a LGS inthat region; namely, in the sampling region, a probing light transmitsthrough, in sequence, the first plate, a sample between the two plates,and the second plate without encountering LGS.

The term “distance between the sampling region and the reference region”of an OAC device can refer to the shortest separation between theboundary of reference region and the boundary of the sampling region.

The terms “imager” and “camera” are interchangeable.

The terms a pillar, a LSG or an object “inside a sample” means that thesidewall of the pillar, the LSG, or the object is surrounded by thesample.unifor

The term “imprinted” means that a spacer and a plate are fixedmonolithically by imprinting (e.g. embossing) a piece of a material toform the spacer on the plate surface. The material can be single layerof a material or multiple layers of the material.

The term “etched” means that a spacer and a plate are fixedmonolithically by etching a piece of a material to form the spacer onthe plate surface. The material can be single layer of a material ormultiple layers of the material.

The term “fused to” means that a spacer and a plate are fixedmonolithically by attaching a spacer and a plate together, the originalmaterials for the spacer and the plate fused into each other, and thereis clear material boundary between the two materials after the fusion.

The term “bonded to” means that a spacer and a plate are fixedmonolithically by binding a spacer and a plate by adhesion.

The term “attached to” means that a spacer and a plate are connectedtogether.

The terms “CROF Card (or card)”, “COF Card”, “QMAX-Card”, “Q-Card”,“CROF device”, “COF device”, “QMAX-device”, “CROF plates”, “COF plates”,and “QMAX-plates” are interchangeable, except that in some embodiments,the COF card does not comprise spacers; and the terms refer to a devicethat comprises a first plate and a second plate that are movablerelative to each other into different configurations (including an openconfiguration and a closed configuration), and that comprises spacers(except some embodiments of the COF card) that regulate the spacingbetween the plates. The term “X-plate” can refer to one of the twoplates in a CROF card, wherein the spacers are fixed to this plate. Moredescriptions of the COF Card, CROF Card, and X-plate are given in theprovisional application Ser. No. 62/456,065, filed on Feb. 7, 2017,which is incorporated herein in its entirety for all purposes.

The term “open configuration” of the two plates in a QMAX process meansa configuration in which the two plates are either partially orcompletely separated apart and the spacing between the plates is notregulated by the spacers

The term “closed configuration” of the two plates in a QMAX processmeans a configuration in which the plates are facing each other, thespacers and a relevant volume of the sample are between the plates, therelevant spacing between the plates, and thus the thickness of therelevant volume of the sample, is regulated by the plates and thespacers, wherein the relevant volume is at least a portion of an entirevolume of the sample.

The term “a sample thickness is regulated by the plate and the spacers”in a QMAX process means that for a give condition of the plates, thesample, the spacer, and the plate compressing method, the thickness ofat least a port of the sample at the closed configuration of the platescan be predetermined from the properties of the spacers and the plate.

The term “inner surface” or “sample surface” of a plate in a QMAX cardcan refer to the surface of the plate that touches the sample, while theother surface (that does not touch the sample) of the plate is termed“outer surface”.

The term “height” or “thickness” of an object in a QMAX process canrefer to, unless specifically stated, the dimension of the object thatis in the direction normal to a surface of the plate. For example,spacer height is the dimension of the spacer in the direction normal toa surface of the plate, and the spacer height and the spacer thicknessmeans the same thing.

The term “area” of an object in a QMAX process can refer to, unlessspecifically stated, the area of the object that is parallel to asurface of the plate. For example, spacer area is the area of the spacerthat is parallel to a surface of the plate.

The term of QMAX card can refer the device that perform a QMAX (e.g.CROF) process on a sample, and have or not have a hinge that connect thetwo plates.

The term “QMAX card with a hinge and “QMAX card” are interchangeable.

The term “angle self-maintain”, “angle self-maintaining”, or “rotationangle self-maintaining” can refer to the property of the hinge, whichsubstantially maintains an angle between the two plates, after anexternal force that moves the plates from an initial angle into theangle is removed from the plates.

The term “a spacer has a predetermined height” and “spacers have apredetermined inter-spacer distance” means, respectively, that the valueof the spacer height and the inter spacer distance is known prior to aQMAX process. It is not predetermined, if the value of the spacer heightand the inter-spacer distance is not known prior to a QMAX process. Forexample, in the case that beads are sprayed on a plate as spacers, wherebeads are landed at random locations of the plate, the inter-spacerdistance is not predetermined. Another example of not predeterminedinter spacer distance is that the spacers moves during a QMAX processes.

The term “a spacer is fixed on its respective plate” in a QMAX processmeans that the spacer is attached to a location of a plate and theattachment to that location is maintained during a QMAX (i.e. thelocation of the spacer on respective plate does not change) process. Anexample of “a spacer is fixed with its respective plate” is that aspacer is monolithically made of one piece of material of the plate, andthe location of the spacer relative to the plate surface does not changeduring the QMAX process. An example of “a spacer is not fixed with itsrespective plate” is that a spacer is glued to a plate by an adhesive,but during a use of the plate, during the QMAX process, the adhesivecannot hold the spacer at its original location on the plate surface andthe spacer moves away from its original location on the plate surface.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible. One skilledartisan will appreciate that the present invention is not limited in itsapplication to the details of construction, the arrangements ofcomponents, category selections, weightings, pre-determined signallimits, or the steps set forth in the description or drawings herein.The invention is capable of other embodiments and of being practiced orbeing carried out in many different ways.

Principles and Certain Examples

One objective of the present invention is related to devices and methodsfor improving optical transmission analysis of a thin layer of a samplesandwiched between containing between two plates, particularly, forgenerating a reference signal that can improve the optical analysis, andfor an application of assaying an analyte in a sample, e.g. hemoglobinin a blood sample.

Certain biological or chemical properties of a sample can be determinedby measuring the absorption coefficient of a thin sample layer, α_(s),in a light transmission experiment through the sample layer. UsingBeer-Lambert's Law, the light absorption coefficient of a thin samplelayer, α_(s), is related to the incident light intensity (i.e. the lightincident to the sample), I_(i), and the transmitted light intensity(i.e. the light goes through the sample), I_(t):

${{OD} = {{\ln \left( \frac{I_{i}}{I_{t}} \right)} = {\alpha_{s}L_{s}}}},$

where L_(s) is the length (i.e. thickness) of the sample layer, and ODis the optical density through the sample layer. The light absorptioncoefficient of a thin sample layer, α_(s), can be related a property ofthe sample. Therefore, using the Beer-Lambert's Law, one can determine aproperty of a sample by measuring the OD of a sample layer.

However, in practice, it is hard to directly measure the intensity ofboth incident light (i.e. the light directly incident to a sample layer)and transmitted light (i.e. the light directly transmitted through thesample). Typically, what is measured in experiments are the total lighttransmission through both the sample and the sample holder (This isbecause a thin layer sample often needs a sample holder for ameasurement, and the light being measured also goes through the sampleholder). Therefore, there is a need to separate/determine the OD of asample from the total light transmission.

According to the present invention, a particular sample holder, termedOAC (i.e. optical analysis card), is provided, and an optical density ofa material is determined by taking a ratio of the intensities of twotransmitted lights: one is the light that transmits through the samplingregion of the sample holder, and the other is the light that transmitsthrough the reference region of the sample holder, wherein the OD of thesample is determined without directly measuring the incident light.

Sample Holders

According to the present invention, as shown in FIGS. 1 and 2, oneembodiment of a sample holder, termed OAC (i.e. optical analysis card),for analyzing an analyte in a sample (e.g. hemoglobin in a blood sample)by optical transmission using light, comprising:

a first plate, a second plate, a light guiding spacer (LGS), a samplingregion, and a reference region, wherein:

-   -   (i) the first plate and second plate are configured to sandwich        a sample, this is for an optical transmission analysis by light,        into a thin layer between the plates, and each plate has a        sample contact area on its inner surface that contacts the        sample;    -   (ii) the light-guiding spacer (LGS) has a pillar shape, is        sandwiched between the two plates with each end of the pillar in        direct contact to one of the plates forming a LGS-plate contact        area, and is configured to allow the light transmits from the        first plate, through the LGS, to the second plate without going        through a sample,    -   (iii) the sampling region is the region that the light can go        through, in sequence, the first plate, the sample, and the        second plate, wherein the sampling region does not have the LGS;        and    -   (iv) the reference region is the region that the light transmits        through, in sequence, the first plate, the light-guiding spacer,        and the second plate, without going through the sample;

wherein the LGS-contact areas and a lateral cross-section of the LGS arelarger than the wavelength of the light,

wherein the light-guiding spacer is surrounded by or near the sample;and

wherein the sample in the sampling region has a thickness of 500 um orless.

At least a portion of the plates in the reference region and thesampling region are light transmissive.

According to the present invention, as shown in FIGS. 1 and 2, a sampleholder, termed OAC (i.e. optical analysis card), has at least a“sampling region” and a “reference region”, and the sample layer lightabsorption coefficient is determined by taking a ratio of the lighttransmitted through the sampling region to that transmitted through thereference region.

In some embodiments, the sample holder (also termed device) furthercomprises a plurality of light guiding spacers, that have substantiallyuniform height, and wherein at least one of the light-guiding spacers isinside the sample contact area.

In some embodiments, the first plate and the second plate are fixed withthe LGS (FIG. 1A). In some embodiments, as shown in FIG. 1B, the firstplate and the second plate are movable relative to each other intodifferent configurations including an open configuration and a closedconfiguration. In an open configuration, the plates are separated a partand a sample is deposited. In a closed configuration, the first andsecond plate is respectively in touch with a flat end of the LGS.

In some embodiments, the first plate and the second plate in the sampleregions and the references have uniform thickness, and are lighttransmissive.

The materials of the plates are plastics, glass, or other materialsdescribed by the disclosure.

In some embodiments, other spacers are used to regulate the spacingbetween the first plate and the second plate, and hence the samplethickness.

Sample OD Measurement Methods.

According to the present invention, a properties of the sample isdetermined by measuring the OD of a thin layer of the sample, where theOD is determined from the ratio of the light transmitted through thesampling region of OAC to that transmitted through the reference regionof OAC.

In some embodiments, the image of a sample in a sample holder is take bya camera and analyzed. (e.g. FIG. 5)

In some embodiments, the wavelength of the light is in the range of 500nm to 1200 nm, 200 nm to 3000 nm, 3000 nm to 30,000 nm, or 100 nm to 200nm.

A) Light Absorption Through a Sample Determined by Light Transmissionsin Sampling and Reference Regions

For the light with an incident light intensity, I₀, the transmittedlight intensity through a sample, I_(s) is, using Beer-Lambert's Law,given by:

${{OD}_{s} = {{\ln \left( \frac{I_{0}}{I_{s}} \right)} = {ɛ_{s}{cL}_{s}}}},$

where, ε_(s) is the extinction coefficient of the sample (e.g.hemoglobin), c is the average concentration of the sample (e.g.hemoglobin), and L is the length of light path through the sample. (ε incm⁻¹/M, c in M, L in cm), and OD_(s) can refer to the optical densitythrough sample.

For the light with an incident light intensity, I₀, the transmittedlight intensity through a light-guiding spacer of a length L_(r), I_(r)is, using Beer-Lambert's Law, given by:

${{OD}_{r} = {{\ln \left( \frac{I_{0}}{I_{r}} \right)} = {\alpha_{r}L_{r}}}},$

where α_(r) is the absorption coefficient of the light-guiding spacer,and L is the length of light path through the sample, and OD_(s) canrefer to the optical density through the light guiding spacer, which isused as a reference.

Subtracting the first equation by the second equation leads to:

${{OD}_{s} - {OD}_{r}} = {{{\ln \left( \frac{I_{0}}{I_{s}} \right)} - {\ln \left( \frac{I_{0}}{I_{r}} \right)}} = {{\ln \left( \frac{I_{r}}{I_{s}} \right)} = {{ɛ_{s}{cL}_{s}} - {\alpha_{r}L_{r}}}}}$

According to the present invention, the above equation shows that theabsorption coefficient of a sample layer can be determined by taking aratio of the transmitted light through the sampling region to thatthrough the reference region, without measuring the incident light(assuming the incident light in the two regions are significantly thesame).

Forming Uniform Thin Sample Layer Using Spacers.

FIG. 1 is an illustration of a CROF (Compressed Regulated Open Flow)embodiment. Panel (a) illustrates a first plate and a second platewherein the first plate has spacers. Panel (b) illustrates depositing asample on the first plate (shown), or the second plate (not shown), orboth (not shown) at an open configuration. Panel (c) illustrates (i)using the two plates to spread the sample (the sample flow between theplates) and reduce the sample thickness, and (ii) using the spacers andthe plate to regulate the sample thickness at the closed configuration.The inner surface of each plate may have one or a plurality of bindingsites and or storage sites (not shown).

B) Two Kinds of Hemoglobin

There are two kinds of hemoglobin in blood. Oxygenated hemoglobin [HbO₂]is the form of hemoglobin with the bound oxygen while deoxygenatedhemoglobin [Hb] is the form of hemoglobin without the bound oxygen.Typically, oxygenated hemoglobin [HbO₂] is around 75% in vein and 90% inartery.

Total hemoglobin concentration=[HbO₂]+[Hb].

Two kinds of hemoglobin can have different extinction coefficient (i.e.light absorption) at different wavelengths as shown in FIG. 4.Therefore, by measuring the light absorption of blood over a differentwavelength range, the concentration of the [HbO₂] and [Hb] in the bloodcan be determined, respectively.

C) Optical Transmission Sample Analysis by Comparing the LightTransmission from the Sampling Region and from the Reference Region

According to the present invention, the light absorption (and opticaldensity (“OD”)) through a thin sample layer is measured by comparing thelight transmission from the sample region and from the reference region.

In some cases, the comparison is taking ratio of the light transmissionfrom the sample region to the reference region.

D) Improved Optical Transmission Sample Analysis

In many real measurement situations, there are many imperfections thatcan significantly reduce the accuracy of OD measurements. For examples,the sample in a sample holder and/or the sample holder itself can have anon-uniform thickness. There are defects in the sample or sample holder,such as air bubbles, dust, or others that can an optical transmissiondifferent from that through a perfect (i.e. ideal sample). The lightintensity may not be uniform in the entire measurement area.

The present invention has a number of ways to reduce errors in anoptical transmission sample analysis (OTSA) caused by the imperfection.According to the present invention, to improve the OD measurementaccuracy, the following features, devices and methods below (i.e. insection 1.4 and its subsection) are used individually or a combinationof thereof.

I. Reduction of Light Scattering by LGS Sidewall and/or LGS-PlateInterface

According to the present invention, in one of the embodiments of the ODmeasurement methods that measures the light intensity of the sampleregion and the reference region, and then takes a ratio of the twointensities, the measurement accuracy can be significantly reduced ifthe light that goes through the reference region has a strong scatteringfrom (a) the LGS sidewall or (b) the LGS, or the light that goes fromthe sample region has a significant scattering from a nearby LGSsidewall.

To reduce the effects of the light scatting by the LGS sidewall on thelight from the reference region, the edge of the reference region usedfor OD determination should be certain distance away from the LGSsidewall. Since the reference region cannot smaller than that of thewavelength of the light without suffering significant light diffraction,therefore to reduce the effects of the light scatting by the LGSsidewall on the light from the reference region, at least thecross-section of LGS should be larger than the wavelength of the light.

In some embodiments, the edge of the reference region used for ODdetermination is certain distance away from the LGS sidewall.

In some embodiments, the cross-section of LGS should be larger than thewavelength of the light, and the edge of the reference region used forOD determination is certain distance away from the LGS sidewall.

Similar to the light from the reference region, to reduce the effects ofthe light scattering on the light from the sampling region, the edge ofthe sampling region should be a certain distance away from the LGSsidewall.

In some embodiments, the edge of the sampling region used for ODdetermination is certain distance away from the LGS sidewall.

In some embodiments, the edge of the reference region used for ODdetermination is certain distance away from the LGS sidewall, and theedge of the sampling region used for OD determination is certaindistance away from the LGS sidewall.

In some embodiments, the cross-section of LGS should be larger than thewavelength of the light, the edge of the reference region used for ODdetermination is certain distance away from the LGS sidewall, and theedge of the sampling region used for OD determination is certaindistance away from the LGS sidewall.

II. Areas of Reference Region and Sampling Region, and Distance Betweenthem

In determining an OD of a sample by taking the ratio of the lightintensities through the sample region and through the reference region,it assumes that the incident light in each region has the sameintensity, or the thickness of the first plate and the second plate andthe sample is respectively the same or known in the sampling region andthe reference region. However, in many practical optical systems,neither of the above assumption is true, which causes uncertainties(i.e. errors) in determining the OD. For examples, in practice, theintensity of incident light for a sample optical transmissionmeasurement is not uniform, particularly illumination area is large; andthe thickness of the first plate, the second plate, and the sample isrespectively not the same or known in the sampling region and thereference region, and each may have a significant variation.

According to the present invention, one way to reduce to errors is tolimit the areas of the sampling region and the reference region used todetermining an OD of the sample, or make the distance between thesampling region and the reference region small while avoiding the lightscattering by the LGS sidewall, or both.

In some embodiments, the area of the sampling region and the distancebetween the sample region and the reference region are a combination ofthe above the two paragraphs.

III. Multiple Pairs of Sampling Region and Reference Region

Using one pair of sample region and the reference region can lead to alarge error. This is because several reasons: (i) since the spatialvariation of the thickness of the first plate, the second plate, and thesample is respectively random, just one pair of sample region andreference region may not represent the majority of the sample; and (ii)since the numbers of optical imperfection and their locations are alsorandom, these optical imperfection can occur at the location of thesampling region and/or the reference region, making the sampling regionand the reference region pair unusable in OTSA.

To solve these problems, according to the present invention, multiplepairs of the SR regions are used.

In some embodiments, an OAC comprises a plurality of pairs of SRregions, where the distance between the centers of two neighboring SRregions, and the distance is either substantially periodic or aperiodic.

According to the present invention, reagents for facilitating a testwere deposited on the inner surface of the plates of an OAC, thereagents include but not limited to staining reagents, surfactants,antibodies, proteins, and nucleic acids.

Light Guiding Pillar (e.g., Spacer)

In some embodiments, the light guiding spacers are substantiallyperiodic in inter spacer distance and predetermined.

Height of the Light Guiding Pillar

In some embodiments, the height of light-guiding spacer is 1 um, 2 um, 5um, 10 um, 30 um, 50 um, 100 um, 200 um, 500 um, 1,000 um, 2,000 um,5,000 um, 10,000 um, or in a range between any of the two values.

Periodicity of the Light Guiding Pillar

In some embodiments, spacer are arranged in periodic array with a periodof 1 um, 2 um, 5 um, 10 um, 30 um, 50 um, 100 um, 200 um, 500 um, 1,000um, 2,000 um, 5,000 um, 10,000 um, or in a range between any of the twovalues.

Geometry of Light Guiding Spacers (LGS)

In some embodiments, the LGS has a pillar shape with its endssubstantially flat. In some embodiments, one or both of the ends of theLGS are fixed with one or both of the plates by bonding, fusing, madefrom a single piece, or other methods that connect LGS to the plates.

In some embodiments, the shape of the lateral cross-section of LGSincludes, not limited to circular, rectangle, square, triangle, polygon,alphabets, numbers, or a combination of thereof.

In some embodiments, the average lateral cross-section of eachlight-guiding spacer (LGS) is 1 um{circumflex over ( )}2(micron-square), 10 um{circumflex over ( )}2, 20 um{circumflex over( )}2, 30 um{circumflex over ( )}2, 50 um{circumflex over ( )}2, 100um{circumflex over ( )}2, 150 um{circumflex over ( )}2, 200um{circumflex over ( )}2, 300 um{circumflex over ( )}2, 500um{circumflex over ( )}2, 1000 um{circumflex over ( )}2, 2000um{circumflex over ( )}2, 5000 um{circumflex over ( )}2, 10,000um{circumflex over ( )}2, 30,000 um{circumflex over ( )}2, 100,000um{circumflex over ( )}2, 200,000 um{circumflex over ( )}2, 500,000um{circumflex over ( )}2, 1 mm{circumflex over ( )}2, 2 mm{circumflexover ( )}2, 5 mm{circumflex over ( )}2, 10 mm{circumflex over ( )}2, 50mm{circumflex over ( )}2, or in a range between any of the two values.

In some preferred embodiments, in the average lateral cross-section ofeach light-guiding spacer is 1 um{circumflex over ( )}2 (micron-square),10 um{circumflex over ( )}2, 20 um{circumflex over ( )}2, 30um{circumflex over ( )}2, 50 um{circumflex over ( )}2, 100 um{circumflexover ( )}2, 150 um{circumflex over ( )}2, 200 um{circumflex over ( )}2,300 um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, 100,000 um{circumflex over ( )}2, 200,000um{circumflex over ( )}2, or in a range between any of the two values.

In certain preferred embodiments, the average lateral cross-section ofeach light-guiding spacer is 1 um{circumflex over ( )}2 (micron-square),10 um{circumflex over ( )}2, 20 um{circumflex over ( )}2, 30um{circumflex over ( )}2, 50 um{circumflex over ( )}2, 100 um{circumflexover ( )}2, 150 um{circumflex over ( )}2, 200 um{circumflex over ( )}2,300 um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, or in a range between any of the two values.

In certain preferred embodiments, the average lateral cross-section ofeach light-guiding spacer is 1 um{circumflex over ( )}2 (micron-square),10 um{circumflex over ( )}2, 20 um{circumflex over ( )}2, 30um{circumflex over ( )}2, 50 um{circumflex over ( )}2, 100 um{circumflexover ( )}2, 150 um{circumflex over ( )}2, 200 um{circumflex over ( )}2,300 um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, or in a range between any of the two values.

In some embodiments, the average lateral cross-section of eachlight-guiding spacer is larger than the wavelength of the light thatgoes through the reference region, by 1 fold, 2 fold, 3 fold, 5 fold, 10fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000fold or in a range between any of the two values.

In certain preferred embodiments, the average lateral cross-section ofeach light-guiding spacer is larger than the wavelength of the lightthat goes through the reference region, by 1 fold, 2 fold, 3 fold, 5fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, or in arange between any of the two values.

Reference Region Geometry

Shape

In some embodiments, the reference region is less than the size of theminimum lateral cross-section of the light guiding pillar. One advantageis to avoid or reduce light scattering the light guiding sidewall toaffect the reference signal.

In some embodiments, the minimum distance between the edge of the lightguiding spacer and that of the reference region is 1 um (micron), 2 um,3 um, 5 um, 10 um, 20 um, 30 um, 50 um, 100 um, 200 um, 500 um, 1000 um,or in a range between any of the two values.

In some preferred embodiments, the minimum distance between the edge ofthe light guiding spacer and that of the reference region is 1 um(micron), 2 um, 3 um, 5 um, 10 um, 20 um, 30 um, 50 um, 100 um, 200 um,or in a range between any of the two values.

In certain preferred embodiments, the minimum distance between the edgeof the light guiding spacer and that of the reference region is 1 um(micron), 2 um, 3 um, 5 um, 10 um, 20 um, 30 um, 50 um, or in a rangebetween any of the two values.

In certain preferred embodiments, the minimum distance between the edgeof the light guiding spacer and that of the reference region is largerthan the wavelength, that goes through the reference region, by 1 fold,2 fold, 3 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold,500 fold, 1000 fold, 5000 fold or in a range between any of the twovalues.

In certain preferred embodiments, the minimum distance between the edgeof the light guiding spacer and that of the reference region is largerthan the wavelength, that goes through the sampling region, by 1 fold, 2fold, 3 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500fold, 1000 fold, 5000 fold or in a range between any of the two values.

The ratio of the reference region area and the light guiding spacer areais 3/10, 2/5, 1/2, 3/5, 7/10, 4/5, or in a range between any of the twovalues.

Sampling Region Geometry

In some embodiments, the edge of the sampling region is a distance awayfrom the edge of the light guiding pillar. One advantage is to avoid orreduce light scattering the light guiding sidewall to affect thereference signal.

In some preferred embodiments, the area of the sampling region is 3/5,7/10, 4/5, 9/10, 1, 11/10, 6/5, 13/10, 7/5, 3/2, or in the range betweenany of the two values, of the periodic inter spacer distance.

In some preferred embodiments, the distance between the edge of thesampling region and that of the light guiding spacer is 1/5, 3/10, 2/5,1/2, 3/5, 7/10, 4/5, 9/10, 1, or in the rage between any of the twovalues, of the light guiding spacer area.

In some preferred embodiments, the distance between the edge of thesampling region and that of the light guiding spacer is larger than thewavelength, that goes through the reference region, by 1 fold, 2 fold, 3fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold,1000 fold, 5000 fold or in a range between any of the two values.

In some preferred embodiments, the distance between the edge of thesampling region and that of the light guiding spacer is larger than thewavelength, that goes through the sampling region, by 1 fold, 2 fold, 3fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold,1000 fold, 5000 fold or in a range between any of the two values.

Distance Between Sampling Region and Reference Region

In some embodiments, the distance between the edge of sampling area andthe reference region is 1 um (micron), 2 um, 3 um, 5 um, 10 um, 20 um,30 um, 40 um, 50 um, 100 um, 200 um, 500 um, 1000 um or in the rangebetween any of the two values.

In certain preferred embodiments, the distance between the edge ofsampling area and the reference region is from 30 um (micron) to 50 um,20 um to 60 um, 10 um to 70 um, 5 um to 75 um, or in the range betweenany of the two values.

In certain preferred embodiments, the distance between the edge ofsampling area and the reference region is larger than the wavelength,that goes through the reference region, by 1 fold, 2 fold, 3 fold, 5fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000fold, 5000 fold or in a range between any of the two values.

In some embodiments, the distance between the edge of sampling area andthe reference region is 2/5, 1/2, 3/5, 7/10, 4/5, 9/10, 1, 11/10, 6/5,13/10, 7/5, 3/2, 8/5, 17/10, or in the range between any of the twovalues, of the light guiding spacer area.

In some embodiments, the distance between the edge of sampling area andthe reference region is larger than the wavelength of the light thatgoes through the reference region, by 1 fold, 2 fold, 3 fold, 5 fold, 10fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000fold or in a range between any of the two values.

In some embodiments, the distance between the edge of sampling area andthe reference region is larger than the wavelength, that goes throughthe sampling region, by 1 fold, 2 fold, 3 fold, 5 fold, 10 fold, 20fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000 fold or ina range between any of the two values.

Lysing Reagent Coating for iMOST-HgB Device

In some embodiment, surfactant is coated on the plate and dissolved intothe blood to achieve a uniform distribution of red blood cell in device,wherein the coating can be on first plate, or second plate, or both.

In some embodiment, surfactant is coated on the plate and dissolved intothe blood to lyse the red blood cell in device, wherein the coating canbe on first plate, or second plate, or both.

In some embodiment, the surfactant coated in the device including butnot limit to Zwittergent, ASB-14, ASB-16, CHAPS, Cationic surfactantNN-[Tris(hydroxymethyl) methyl]-N-alkyl-N,N-dimethyl ammonium chloride(IIa), IIb, IIc, IId, CTAC, Tween 20, Tween 40, Tween 60, Tween 80,Sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodiumlauryl ether sulfate (SLES), sodium myreth sulfate, docusate,perfluorooctanesulfonate, alkyl-aryl ether phosphates, alkyl etherphosphates, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride(BAC), benzethonium chloride (BZT), dimethyldioctadecylammoniumchloride, dioctadecyldimethlyammonium bromide (DODAB), cocamidopropylhydroxysultaine, cocamidopropyl betaine, narrow-range ethoxylate,octaethylene glycol monododecyl ether, pentaethylene glycol monododecylether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamidemonoethanolamine, cocamide diethanolamine, poloxamers, glycerolmonostearate, glycerol monolaurate, sorbitan monolaurate, sorbitanmonostearate, sorbitan tristearate, decyl glucoside, lauryl glucoside,octyl glucoside, lauryldimethylamine oxide, dimethyl sulfoxide,phosphine oxide.

In some embodiment, the reagent causing red blood cell lysis coated inthe device including but not limit to Pluronic F-127, Cremophor EL,Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium dodecyl sulfate,Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, Tamoxifen,saponin, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid,lactic acid, ABS-14, ABS-16, anti-malaria drugs (quinine compounds),arsenic, dapsone, metals (chromium/chromates, platinum salts, nickelcompounds, copper, lead, cis-platinum), nitrites, nitrofurantoin,penicillin, phenazopyridine (pyridium), rho immune globulin, ribavirin,sulfonamides, sulfones.

In some embodiment, the anticoagulant coated in the device including butnot limit to EDTA such as dipotassium ethylenediaminetetraacetic acid(K2EDTA), tripotassium ethylenediaminetetraacetic (K3EDTA), coumarins(vitamin K antagonists), warfarin (coumadin), acenocoumarol,phenprocoumon, atromentin, phenindione, heparin, fondaparinux andidraparinux, dabigatran, rivaroxaban, apixaban, edoxaban, betrixaban,NOACs, hirudin, lepirudin, bivalirudin, agratroban, dabigatran,batroxobin, hementin, Vitamin E, sodium citrate, acid citrate dextrose,oxalate such as fluoride oxalate, deltaparin, desirudin, enoxaparin.

In some embodiment, to achieve a uniform distribution of red blood cellin device, Zwittergent is coated on the plate with a preferred areaconcentration of 3 ng/mm², 5 ng/mm², 8 ng/mm², 12 ng/mm², 15 ng/mm², 25ng/mm², 35 ng/mm², 50 ng/mm², 80 ng/mm², 100 ng/mm² or in a rangebetween any of the two values.

In some embodiment, to lyse red blood cell in device, Zwittergent iscoated on the plate with a preferred area concentration of 100 ng/mm²,120 ng/mm², 150 ng/mm², 180 ng/mm², 200 ng/mm², 300 ng/mm², 400 ng/mm²,500 ng/mm², 800 ng/mm², 1000 ng/mm² or in a range between any of the twovalues.

In some embodiment, to achieve a uniform distribution of red blood cellin device, Zwittergent is coated on the plate with a preferred finalconcentration in blood of 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL,0.5 mg/mL, 0.6 mg/mL, 1.0 mg/mL, 2 mg/mL or in a range between any ofthe two values.

In some embodiment, to lyse red blood cell in device, Zwittergent iscoated on the plate with a preferred final concentration in blood of 2mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 9 mg/mL, 10 mg/mL,15 mg/mL, 25 mg/mL, 50 mg/mL, or in a range between any of the twovalues.

Measurement Range for iMOST-HgB Device

In some embodiment, the hemoglobin measurement range of the device isbetween 0 g/dL to 40 g/dL.

In some embodiment, the preferred hemoglobin measurement range of thedevice is between 0 g/dL to 30 g/dL.

In some embodiment, the preferred hemoglobin measurement range of thedevice is between 5 g/dL to 26 g/dL.

Scattering Particle Removal and Compensation

In certain situations, there are imperfections that can significantlyreduce the accuracy of absorption and/or hemoglobin measurements.

For examples, there are interfering particles in the sample or sampleholder, including but not limited to, particles increasing turbidity,light scattering particle, air bubbles, dust, or others that can anoptical transmission different from that through a perfect (i.e. idealsample).

The present invention has a number of ways to reduce errors in anoptical transmission sample analysis (OTSA) caused by the imperfection.

In some embodiment, multiple pairs of the SR regions are used. For eachpair of the SR region, an OD of a sample is determined by taking theratio of the light intensities through the sample region and through thereference region. For a given pair of the SR region, a qualitymeasurement of the SR region is calculated; If the quality measurementis low, this pair of the SR region is excluded from the poolingalgorithm in the next stage. The pooling algorithm is to pool OD of asample over all pairs of SR regions. Various pooling algorithm can beutilized, including but not limited to median, mean, max, min, k-means,etc.

In some embodiment, the imperfection areas are removed or excluded fromthe image before the light intensities analyze.

In some embodiment, the imperfection areas with a boundary are removedor excluded from the image before the light intensities analyze, whereinthe boundary size is between 1 um to 50 um; wherein the preferredboundary size is between 5 um to 20 um.

Measurement with/without Scanning

In certain embodiment, one location on device is measured for analysis,particularly for Hemoglobin.

QMAX System A) QMAX Card

Details of the QMAX card are described in detail in a variety ofpublications including International Application No. PCT/US2016/046437(Essenlix Docket No. ESSN-028WO), which is hereby incorporated byreference herein for all purposes.

I. Plates

In present invention, generally, the plates of CROF are made of anymaterial that (i) is capable of being used to regulate, together withthe spacers, the thickness of a portion or entire volume of the sample,and (ii) has no significant adverse effects to a sample, an assay, or agoal that the plates intend to accomplish. However, in certainembodiments, particular materials (hence their properties) are used forthe plate to achieve certain objectives.

In certain embodiments, the two plates have the same or differentparameters for each of the following parameters: plate material, platethickness, plate shape, plate area, plate flexibility, plate surfaceproperty, and plate optical transparency.

(i) Plate Materials.

The plates are made a single material, composite materials, multiplematerials, multilayer of materials, alloys, or a combination thereof.Each of the materials for the plate is an inorganic material, am organicmaterial, or a mix, wherein examples of the materials are given inparagraphs of Mat-1 and Mat-2.

Mat-1: The inorganic materials for the plates include, not limited to,glass, quartz, oxides, silicon-dioxide, silicon-nitride, hafnium oxide(HfO), aluminum oxide (AlO), semiconductors: (silicon, GaAs, GaN, etc.),metals (e.g. gold, silver, copper, aluminum, Ti, Ni, etc.), ceramics, orany combinations of thereof.

Mat-2: The organic materials for the spacers include, not limited to,polymers (e.g. plastics) or amorphous organic materials. The polymermaterials for the spacers include, not limited to, acrylate polymers,vinyl polymers, olefin polymers, cellulosic polymers, noncellulosicpolymers, polyester polymers, Nylon, cyclic olefin copolymer (COC),poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefinpolymer (COP), liquid crystalline polymer (LCP), polyimide (PA),polyethylene (PE), polyimide (PI), polypropylene (PP), poly(phenyleneether) (PPE), polystyrene (PS), polyoxymethylene (POM), polyether etherketone (PEEK), polyether sulfone (PES), poly(ethylene phthalate) (PET),polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidenefluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane(PDMS), rubbers, or any combinations of thereof.

In certain embodiments, the plates are each independently made of atleast one of glass, plastic, ceramic, and metal. In certain embodiments,each plate independently includes at least one of glass, plastic,ceramic, and metal.

In certain embodiments, one plate is different from the other plate inlateral area, thickness, shape, materials, or surface treatment. Incertain embodiments, one plate is the same as the other plate in lateralarea, thickness, shape, materials, or surface treatment.

The materials for the plates are rigid, flexible or any flexibilitybetween the two. The rigid (e.g. stiff) or flexibility is relative to agive pressing forces used in bringing the plates into the closedconfiguration.

In certain embodiments, a selection of rigid or flexible plate aredetermined from the requirements of controlling a uniformity of thesample thickness at the closed configuration.

In certain embodiments, at least one of the two plates are transparent(to a light). In certain embodiments at least a part or several parts ofone plate or both plates are transparent. In certain embodiments, theplates are non-transparent.

(ii) Plate Thickness.

In certain embodiments, the average thicknesses for at least one of theplates are 2 nm or less, 10 nm or less, 100 nm or less, 500 nm or less,1000 nm or less, 2 um (micron) or less, 5 um or less, 10 um or less, 20um or less, 50 um or less, 100 um or less, 150 um or less, 200 um orless, 300 um or less, 500 um or less, 800 um or less, 1 mm (millimeter)or less, 2 mm or less, 3 mm or less, or a range between any two of thevalues.

In certain embodiments, the average thicknesses for at least one of theplates are at most 3 mm (millimeter), at most 5 mm, at most 10 mm, atmost 20 mm, at most 50 mm, at most 100 mm, at most 500 mm, or a rangebetween any two of the values.

In certain embodiments, the thickness of a plate is not uniform acrossthe plate. Using a different plate thickness at different location canbe used to control the plate bending, folding, sample thicknessregulation, and others.

(iii) Plate Shape and Area.

Generally, the plates can have any shapes, as long as the shape allows acompress open flow of the sample and the regulation of the samplethickness. However, in certain embodiments, a particular shape can beadvantageous. The shape of the plate can be round, elliptical,rectangles, triangles, polygons, ring-shaped, or any superpositions ofthese shapes.

In certain embodiments, the two plates can have the same size or shape,or different. The area of the plates depend on the application. The areaof the plate is at most 1 mm2 (millimeter square), at most 10 mm2, atmost 100 mm2, at most 1 cm2 (centimeter square), at most 5 cm2, at most10 cm2, at most 100 cm2, at most 500 cm2, at most 1000 cm2, at most 5000cm2, at most 10,000 cm2, or over 10,000 cm2, or any arrange between anyof the two values. The shape of the plate can be rectangle, square,round, or others.

In certain embodiments, at least one of the plates is in the form of abelt (or strip) that has a width, thickness, and length. The width is atmost 0.1 cm (centimeter), at most 0.5 cm, at most 1 cm, at most 5 cm, atmost 10 cm, at most 50 cm, at most 100 cm, at most 500 cm, at most 1000cm, or a range between any two of the values. The length can be as longit needed. The belt can be rolled into a roll.

(iv) Plate Surface Flatness.

In many embodiments, an inner surface of the plates are flat orsignificantly flat, planar. In certain embodiments, the two innersurfaces are, at the closed configuration, parallel with each other.Flat inner surfaces facilitates a quantification and/or controlling ofthe sample thickness by simply using the predetermined spacer height atthe closed configuration. For non-flat inner surfaces of the plate, oneneed to know not only the spacer height, but also the exact the topologyof the inner surface to quantify and/or control the sample thickness atthe closed configuration. To know the surface topology needs additionalmeasurements and/or corrections, which can be complex, time consuming,and costly.

A flatness of the plate surface is relative to the final samplethickness (the final thickness is the thickness at the closedconfiguration), and is often characterized by the term of “relativesurface flatness” is the ratio of the plate surface flatness variationto the final sample thickness.

In certain embodiments, the relative surface is less than 0.01%, 0.1%,less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%,less than 20%, less than 30%, less than 50%, less than 70%, less than80%, less than 100%, or a range between any two of these values.

(v) Plate Surface Parallelness.

In certain embodiments, the two surfaces of the plate is significantlyparallel with each other. In certain embodiments, the two surfaces ofthe plate is not parallel with each other.

(vi) Plate Flexibility.

In certain embodiments, a plate is flexible under the compressing of aCROF process. In certain embodiments, both plates are flexible under thecompressing of a CROF process. In certain embodiments, a plate is rigidand another plate is flexible under the compressing of a CROF process.In certain embodiments, both plates are rigid. In certain embodiments,both plate are flexible but have different flexibility.

(vii) Plate Optical Transparency.

In certain embodiments, a plate is optical transparent. In certainembodiments, both plates are optical transparent. In certainembodiments, a plate is optical transparent and another plate is opaque.In certain embodiments, both plates are opaque. In certain embodiments,both plate are optical transparent but have different opticaltransparency. The optical transparency of a plate can refer to a part orthe entire area of the plate.

(viii) Surface Wetting Properties.

In certain embodiments, a plate has an inner surface that wets (e.g.contact angle is less 90 degree) the sample, the transfer liquid, orboth. In certain embodiments, both plates have an inner surface thatwets the sample, the transfer liquid, or both; either with the same ordifferent wettability. In certain embodiments, a plate has an innersurface that wets the sample, the transfer liquid, or both; and anotherplate has an inner surface that does not wet (e.g. the contact angleequal to or larger than 90 degree). The wetting of a plate inner surfacecan refer to a part or the entire area of the plate.

In certain embodiments, the inner surface of the plate has other nano ormicrostructures to control a lateral flow of a sample during a CROF. Thenano or microstructures include, but not limited to, channels, pumps,and others. Nano and microstructures are also used to control thewetting properties of an inner surface.

II. Spacers

(i) Spacers' Function.

In present invention, the spacers are configured to have one or anycombinations of the following functions and properties: the spacers areconfigured to (1) control, together with the plates, the thickness ofthe sample or a relevant volume of the sample (Preferably, the thicknesscontrol is precise, or uniform or both, over a relevant area); (2) allowthe sample to have a compressed regulated open flow (CROF) on platesurface; (3) not take significant surface area (volume) in a givensample area (volume); (4) reduce or increase the effect of sedimentationof particles or analytes in the sample; (5) change and/or control thewetting propertied of the inner surface of the plates; (6) identify alocation of the plate, a scale of size, and/or the information relatedto a plate, or (7) do any combination of the above.

(ii) Spacer Architectures and Shapes.

To achieve desired sample thickness reduction and control, in certainembodiments, the spacers are fixed its respective plate. In general, thespacer can have any shape, as long as the spacers are capable ofregulating the sample thickness during a CROF process, but certainshapes are preferred to achieve certain functions, such as betteruniformity, less overshoot in pressing, etc.

The spacer(s) is a single spacer or a plurality of spacers. (e.g. anarray). Certain embodiments of a plurality of spacers is an array ofspacers (e.g. pillars), where the inter-spacer distance is periodic oraperiodic, or is periodic or aperiodic in certain areas of the plates,or has different distances in different areas of the plates.

There are two kinds of the spacers: open-spacers and enclosed-spacers.The open-spacer is the spacer that allows a sample to flow through thespacer (e.g. the sample flows around and pass the spacer. For example, apost as the spacer.), and the enclosed spacer is the spacer that stopthe sample flow (e.g. the sample cannot flow beyond the spacer. Forexample, a ring shape spacer and the sample is inside the ring.). Bothtypes of spacers use their height to regular the final sample thicknessat a closed configuration.

In certain embodiments, the spacers are open-spacers only. In certainembodiments, the spacers are enclosed-spacers only. In certainembodiments, the spacers are a combination of open-spacers andenclosed-spacers.

The term “pillar spacer” means that the spacer has a pillar shape andthe pillar shape can refer to an object that has height and a lateralshape that allow a sample to flow around it during a compressed openflow.

In certain embodiments, the lateral shapes of the pillar spacers are theshape selected from the groups of (i) round, elliptical, rectangles,triangles, polygons, ring-shaped, star-shaped, letter-shaped (e.g.L-shaped, C-shaped, the letters from A to Z), number shaped (e.g. theshapes like 0 1, 2, 3, 4, . . . . to 9); (ii) the shapes in group (i)with at least one rounded corners; (iii) the shape from group (i) withzig-zag or rough edges; and (iv) any superposition of (i), (ii) and(iii). For multiple spacers, different spacers can have differentlateral shape and size and different distance from the neighboringspacers.

In certain embodiments, the spacers can be and/or can include posts,columns, beads, spheres, and/or other suitable geometries. The lateralshape and dimension (e.g., transverse to the respective plate surface)of the spacers can be anything, except, in certain embodiments, thefollowing restrictions: (i) the spacer geometry will not cause asignificant error in measuring the sample thickness and volume; or (ii)the spacer geometry would not prevent the out-flowing of the samplebetween the plates (e.g. it is not in enclosed form). But in certainembodiments, they require some spacers to be closed spacers to restrictthe sample flow.

In certain embodiments, the shapes of the spacers have rounded corners.For example, a rectangle shaped spacer has one, several or all cornersrounded (like a circle rather 90 degree angle). A round corner oftenmake a fabrication of the spacer easier, and in some cases less damageto a biological material.

The sidewall of the pillars can be straight, curved, sloped, ordifferent shaped in different section of the sidewall. In certainembodiments, the spacers are pillars of various lateral shapes,sidewalls, and pillar-height to pillar lateral area ratio. In apreferred embodiment, the spacers have shapes of pillars for allowingopen flow.

(iii) Spacers' Materials.

In the present invention, the spacers are generally made of any materialthat is capable of being used to regulate, together with the two plates,the thickness of a relevant volume of the sample. In certainembodiments, the materials for the spacers are different from that forthe plates. In certain embodiments, the materials for the spaces are atleast the same as a part of the materials for at least one plate.

The spacers are made a single material, composite materials, multiplematerials, multilayer of materials, alloys, or a combination thereof.Each of the materials for the spacers is an inorganic material, amorganic material, or a mix, wherein examples of the materials are givenin paragraphs of Mat-1 and Mat-2. In a preferred embodiment, the spacersare made in the same material as a plate used in CROF.

(iv) Spacers' Mechanical Strength and Flexibility.

In certain embodiments, the mechanical strength of the spacers arestrong enough, so that during the compression and at the closedconfiguration of the plates, the height of the spacers is the same orsignificantly same as that when the plates are in an open configuration.In certain embodiments, the differences of the spacers between the openconfiguration and the closed configuration can be characterized andpredetermined.

The material for the spacers is rigid, flexible or any flexibilitybetween the two. The rigid is relative to a give pressing forces used inbringing the plates into the closed configuration: if the space does notdeform greater than 1% in its height under the pressing force, thespacer material is regarded as rigid, otherwise a flexible. When aspacer is made of material flexible, the final sample thickness at aclosed configuration still can be predetermined from the pressing forceand the mechanical property of the spacer.

(v) Spacers Inside Sample.

To achieve desired sample thickness reduction and control, particularlyto achieve a good sample thickness uniformity, in certain embodiments,the spacers are placed inside the sample, or the relevant volume of thesample. In certain embodiments, there are one or more spacers inside thesample or the relevant volume of the sample, with a proper inter spacerdistance. In certain embodiments, at least one of the spacers is insidethe sample, at least two of the spacers inside the sample or therelevant volume of the sample, or at least of “n” spacers inside thesample or the relevant volume of the sample, where “n” can be determinedby a sample thickness uniformity or a required sample flow propertyduring a CROF.

(vi) Spacer Height.

In certain embodiments, all spacers have the same pre-determined height.In certain embodiments, spacers have different pre-determined height. Incertain embodiments, spacers can be divided into groups or regions,wherein each group or region has its own spacer height. And in certainembodiments, the predetermined height of the spacers is an averageheight of the spacers. In certain embodiments, the spacers haveapproximately the same height. In certain embodiments, a percentage ofnumber of the spacers have the same height.

The height of the spacers is selected by a desired regulated finalsample thickness and the residue sample thickness. The spacer height(the predetermined spacer height) and/or sample thickness is 3 nm orless, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500nm or less, 800 nm or less, 1000 nm or less, 1 um or less, 2 um or less,3 um or less, 5 um or less, 10 um or less, 20 um or less, 30 um or less,50 um or less, 100 um or less, 150 um or less, 200 um or less, 300 um orless, 500 um or less, 800 um or less, 1 mm or less, 2 mm or less, 4 mmor less, or a range between any two of the values.

The spacer height and/or sample thickness is between 1 nm to 100 nm inone preferred embodiment, 100 nm to 500 nm in another preferredembodiment, 500 nm to 1000 nm in a separate preferred embodiment, 1 um(e.g. 1000 nm) to 2 um in another preferred embodiment, 2 um to 3 um ina separate preferred embodiment, 3 um to 5 um in another preferredembodiment, 5 um to 10 um in a separate preferred embodiment, and 10 umto 50 um in another preferred embodiment, 50 um to 100 um in a separatepreferred embodiment.

In certain embodiments, the spacer height and/or sample thickness (i)equal to or slightly larger than the minimum dimension of an analyte, or(ii) equal to or slightly larger than the maximum dimension of ananalyte. The “slightly larger” means that it is about 1% to 5% largerand any number between the two values.

In certain embodiments, the spacer height and/or sample thickness islarger than the minimum dimension of an analyte (e.g. an analyte has ananisotropic shape), but less than the maximum dimension of the analyte.

For example, the red blood cell has a disk shape with a minim dimensionof 2 um (disk thickness) and a maximum dimension of 11 um (a diskdiameter). In an embodiment of the present invention, the spacers isselected to make the inner surface spacing of the plates in a relevantarea to be 2 um (equal to the minimum dimension) in one embodiment, 2.2um in another embodiment, or 3 (50% larger than the minimum dimension)in other embodiment, but less than the maximum dimension of the redblood cell. Such embodiment has certain advantages in blood cellcounting. In one embodiment, for red blood cell counting, by making theinner surface spacing at 2 or 3 um and any number between the twovalues, a undiluted whole blood sample is confined in the spacing, onaverage, each red blood cell (RBC) does not overlap with others,allowing an accurate counting of the red blood cells visually. (Too manyoverlaps between the RBC's can cause serious errors in counting).

In the present invention, in certain embodiments, it uses the plates andthe spacers to regulate not only a thickness of a sample, but also theorientation and/or surface density of the analytes/entity in the samplewhen the plates are at the closed configuration. When the plates are ata closed configuration, a thinner thickness of the sample gives a lessthe analytes/entity per surface area (e.g. less surface concentration).

-   -   (vii) Spacer Lateral Dimension.

For an open-spacer, the lateral dimensions can be characterized by itslateral dimension (sometime being called width) in the x and y—twoorthogonal directions. The lateral dimension of a spacer in eachdirection is the same or different. In certain embodiments, the lateraldimension for each direction (x or y) is . . . .

In certain embodiments, the ratio of the lateral dimensions of x to ydirection is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or a rangebetween any two of the value. In certain embodiments, a different ratiois used to regulate the sample flow direction; the larger the ratio, theflow is along one direction (larger size direction).

In certain embodiments, the different lateral dimensions of the spacersin x and y direction are used as (a) using the spacers as scale-markersto indicate the orientation of the plates, (b) using the spacers tocreate more sample flow in a preferred direction, or both.

In a preferred embodiment, the period, width, and height.

In certain embodiments, all spacers have the same shape and dimensions.In certain embodiments, each of the spacers have different lateraldimensions.

For enclosed-spacers, in certain embodiments, the inner lateral shapeand size are selected based on the total volume of a sample to beenclosed by the enclosed spacer(s), wherein the volume size has beendescribed in the present disclosure; and in certain embodiments, theouter lateral shape and size are selected based on the needed strengthto support the pressure of the liquid against the spacer and thecompress pressure that presses the plates.

(viii) Aspect Ratio of Height to the Average Lateral Dimension of PillarSpacer.

In certain embodiments, the aspect ratio of the height to the averagelateral dimension of the pillar spacer is 100,000, 10,000, 1,000, 100,10, 1, 0.1, 0.01, 0.001, 0.0001, 0, 00001, or a range between any two ofthe values.

(ix) Spacer Height Precisions.

The spacer height should be controlled precisely. The relative precisionof the spacer (e.g. the ratio of the deviation to the desired spacerheight) is 0.001% or less, 0.01% or less, 0.1% or less; 0.5% or less, 1%or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less,20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% orless, 80% or less, 90% or less, 99.9% or less, or a range between any ofthe values.

(x) Inter-Spacer Distance.

The spacers can be a single spacer or a plurality of spacers on theplate or in a relevant area of the sample. In certain embodiments, thespacers on the plates are configured and/or arranged in an array form,and the array is a periodic, non-periodic array or periodic in somelocations of the plate while non-periodic in other locations.

In certain embodiments, the periodic array of the spacers has a latticeof square, rectangle, triangle, hexagon, polygon, or any combinations ofthereof, where a combination means that different locations of a platehas different spacer lattices.

In certain embodiments, the inter-spacer distance of a spacer array isperiodic (e.g. uniform inter-spacer distance) in at least one directionof the array. In certain embodiments, the inter-spacer distance isconfigured to improve the uniformity between the plate spacing at aclosed configuration.

The distance between neighboring spacers (e.g. the inter-spacerdistance) is 1 um or less, 5 um or less, 10 um or less, 20 um or less,30 um or less, 40 um or less, 50 um or less, 60 um or less, 70 um orless, 80 um or less, 90 um or less, 100 um or less, 200 um or less, 300um or less, 400 um or less, or a range between any two of the values.

In certain embodiments, the inter-spacer distance is at 400 or less, 500or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm orless, 10 mm or less, or any range between the values. In certainembodiments, the inter-spacer distance is a 10 mm or less, 20 mm orless, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, orany range between the values.

The distance between neighboring spacers (e.g. the inter-spacerdistance) is selected so that for a given properties of the plates and asample, at the closed-configuration of the plates, the sample thicknessvariation between two neighboring spacers is, in certain embodiments, atmost 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between thevalues; or in certain embodiments, at most 80%, 100%, 200%, 400%, or arange between any two of the values.

Clearly, for maintaining a given sample thickness variation between twoneighboring spacers, when a more flexible plate is used, a closerinter-spacer distance is needed.

Specify the accuracy of the inter spacer distance.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 2 to 4 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 1 um to 100 um.

In a preferred embodiment, the spacer is a periodic square array,wherein the spacer is a pillar that has a height of 4 to 50 um, anaverage lateral dimension of from 5 to 20 um, and inter-spacer spacingof 100 um to 250 um.

The period of spacer array is between 1 nm to 100 nm in one preferredembodiment, 100 nm to 500 nm in another preferred embodiment, 500 nm to1000 nm in a separate preferred embodiment, 1 um (e.g. 1000 nm) to 2 umin another preferred embodiment, 2 um to 3 um in a separate preferredembodiment, 3 um to 5 um in another preferred embodiment, 5 um to 10 umin a separate preferred embodiment, and 10 um to 50 um in anotherpreferred embodiment, 50 um to 100 um in a separate preferredembodiment, 100 um to 175 um in a separate preferred embodiment, and 175um to 300 um in a separate preferred embodiment.

(xi) Spacer Density.

The spacers are arranged on the respective plates at a surface densityof greater than one per um², greater than one per 10 um², greater thanone per 100 um², greater than one per 500 um², greater than one per 1000um², greater than one per 5000 um², greater than one per 0.01 mm²,greater than one per 0.1 mm², greater than one per 1 mm², greater thanone per 5 mm², greater than one per 10 mm², greater than one per 100mm², greater than one per 1000 mm², greater than one per 10000 mm², or arange between any two of the values.

(3) the spacers are configured to not take significant surface area(volume) in a given sample area (volume);

(xii) Ratio of Spacer Volume to Sample Volume.

In many embodiments, the ratio of the spacer volume (e.g. the volume ofthe spacer) to sample volume (e.g. the volume of the sample), and/or theratio of the volume of the spacers that are inside of the relevantvolume of the sample to the relevant volume of the sample are controlledfor achieving certain advantages. The advantages include, but notlimited to, the uniformity of the sample thickness control, theuniformity of analytes, the sample flow properties (e.g. flow speed,flow direction, etc.).

In certain embodiments, the ratio of the spacer volume r) to samplevolume, and/or the ratio of the volume of the spacers that are inside ofthe relevant volume of the sample to the relevant volume of the sampleis less than 100%, at most 99%, at most 70%, at most 50%, at most 30%,at most 10%, at most 5%, at most 3% at most 1%, at most 0.1%, at most0.01%, at most 0.001%, or a range between any of the values.

(xiii) Spacers Fixed to Plates.

The inter spacer distance and the orientation of the spacers, which playa key role in the present invention, are preferably maintained duringthe process of bringing the plates from an open configuration to theclosed configuration, and/or are preferably predetermined before theprocess from an open configuration to a closed configuration.

In certain embodiments of the present disclosure, spacers are fixed onone of the plates before bring the plates to the closed configuration.The term “a spacer is fixed with its respective plate” means that thespacer is attached to a plate and the attachment is maintained during ause of the plate. An example of “a spacer is fixed with its respectiveplate” is that a spacer is monolithically made of one piece of materialof the plate, and the position of the spacer relative to the platesurface does not change. An example of “a spacer is not fixed with itsrespective plate” is that a spacer is glued to a plate by an adhesive,but during a use of the plate, the adhesive cannot hold the spacer atits original location on the plate surface (e.g. the spacer moves awayfrom its original position on the plate surface).

In certain embodiments, at least one of the spacers are fixed to itsrespective plate. In certain embodiments, at two spacers are fixed toits respective plates. In certain embodiments, a majority of the spacersare fixed with their respective plates. In certain embodiments, all ofthe spacers are fixed with their respective plates.

In certain embodiments, a spacer is fixed to a plate monolithically.

In certain embodiments, the spacers are fixed to its respective plate byone or any combination of the following methods and/or configurations:attached to, bonded to, fused to, imprinted, and etched.

The term “imprinted” means that a spacer and a plate are fixedmonolithically by imprinting (e.g. embossing) a piece of a material toform the spacer on the plate surface. The material can be single layerof a material or multiple layers of the material.

The term “etched” means that a spacer and a plate are fixedmonolithically by etching a piece of a material to form the spacer onthe plate surface. The material can be single layer of a material ormultiple layers of the material.

The term “fused to” means that a spacer and a plate are fixedmonolithically by attaching a spacer and a plate together, the originalmaterials for the spacer and the plate fused into each other, and thereis clear material boundary between the two materials after the fusion.

The term “bonded to” means that a spacer and a plate are fixedmonolithically by binding a spacer and a plate by adhesion.

The term “attached to” means that a spacer and a plate are connectedtogether.

In certain embodiments, the spacers and the plate are made in the samematerials. In other embodiment, the spacers and the plate are made fromdifferent materials. In other embodiment, the spacer and the plate areformed in one piece. In other embodiment, the spacer has one end fixedto its respective plate, while the end is open for accommodatingdifferent configurations of the two plates.

In other embodiment, each of the spacers independently is at least oneof attached to, bonded to, fused to, imprinted in, and etched in therespective plate. The term “independently” means that one spacer isfixed with its respective plate by a same or a different method that isselected from the methods of attached to, bonded to, fused to, imprintedin, and etched in the respective plate.

In certain embodiments, at least a distance between two spacers ispredetermined (“predetermined inter-spacer distance” means that thedistance is known when a user uses the plates.).

In certain embodiments of all methods and devices described herein,there are additional spacers besides to the fixed spacers.

(xiv) Specific Sample Thickness.

In present invention, it was observed that a larger plate holding force(e.g. the force that holds the two plates together) can be achieved byusing a smaller plate spacing (for a given sample area), or a largersample area (for a given plate-spacing), or both.

In certain embodiments, at least one of the plates is transparent in aregion encompassing the relevant area, each plate has an inner surfaceconfigured to contact the sample in the closed configuration; the innersurfaces of the plates are substantially parallel with each other, inthe closed configuration; the inner surfaces of the plates aresubstantially planar, except the locations that have the spacers; or anycombination of thereof.

The spacers can be fabricated on a plate in a variety of ways, usinglithography, etching, embossing (nanoimprint), depositions, lift-off,fusing, or a combination of thereof. In certain embodiments, the spacersare directly embossed or imprinted on the plates. In certainembodiments, the spacers imprinted into a material (e.g. plastics) thatis deposited on the plates. In certain embodiments, the spacers are madeby directly embossing a surface of a CROF plate. The nanoimprinting canbe done by roll to roll technology using a roller imprinter, or roll toa planar nanoimprint. Such process has a great economic advantage andhence lowering the cost.

In certain embodiments, the spacers are deposited on the plates. Thedeposition can be evaporation, pasting, or a lift-off. In the pasting,the spacer is fabricated first on a carrier, then the spacer istransferred from the carrier to the plate. In the lift-off, a removablematerial is first deposited on the plate and holes are created in thematerial; the hole bottom expose the plate surface and then a spacermaterial is deposited into the hole and afterwards the removablematerial is removed, leaving only the spacers on the plate surface. Incertain embodiments, the spacers deposited on the plate are fused withthe plate. In certain embodiments, the spacer and the plates arefabricated in a single process. The single process includes imprinting(e.g. embossing, molding) or synthesis.

In certain embodiments, at least two of the spacers are fixed to therespective plate by different fabrication methods, and optionallywherein the different fabrication methods include at least one of beingdeposition, bonded, fuse, imprinted, and etched.

In certain embodiments, one or more of the spacers are fixed to therespective plate(s) is by a fabrication method of being bonded, beingfused, being imprinted, or being etched, or any combination of thereof.

In certain embodiments, the fabrication methods for forming suchmonolithic spacers on the plate include a method of being bonded, beingfused, being imprinted, or being etched, or any combination of thereof.

B) Adaptor

Details of the Adaptor are described in detail in a variety ofpublications including International Application No. PCT/US2018/017504(Essenlix Docket No. ESXPCT18F04), which is hereby incorporated byreference herein for all purposes.

The present invention that is described herein address this problem byproviding a system comprising an optical adaptor and a smartphone. Theoptical adaptor device fits over a smartphone converting it into amicroscope which can take both fluorescent and bright-field images of asample. This system can be operated conveniently and reliably by acommon person at any location. The optical adaptor takes advantage ofthe existing resources of the smartphone, including camera, lightsource, processor and display screen, which provides a low-cost solutionlet the user to do bright-field and fluorescent microscopy.

In this invention, the optical adaptor device comprises a holder framefitting over the upper part of the smartphone and an optical boxattached to the holder having sample receptacle slot and illuminationoptics. In some references (U.S. Pat. No. 2016/029091 and U.S. Pat. No.2011/0292198), their optical adaptor design is a whole piece includingboth the clip-on mechanics parts to fit over the smartphone and thefunctional optics elements. This design has the problem that they needto redesign the whole-piece optical adaptor for each specific model ofsmartphone. But in this present invention, the optical adaptor isseparated into a holder frame only for fitting a smartphone and auniversal optical box containing all the functional parts. For thesmartphones with different dimensions, as long as the relative positionsof the camera and the light source are the same, only the holder frameneed to be redesigned, which will save a lot of cost of design andmanufacture.

The optical box of the optical adaptor comprises: a receptacle slotwhich receives and position the sample in a sample slide in the field ofview and focal range of the smartphone camera; a bright-fieldillumination optics for capturing bright-field microscopy images of asample; a fluorescent illumination optics for capturing fluorescentmicroscopy images of a sample; a lever to switch between bright-fieldillumination optics and fluorescent illumination optics by slidinginward and outward in the optical box.

The receptacle slot has a rubber door attached to it, which can fullycover the slot to prevent the ambient light getting into the optical boxto be collected by the camera. In U.S. Pat. 2016/0290916, the sampleslot is always exposed to the ambient light which won't cause too muchproblem because it only does bright-field microscopy. But the presentinvention can take the advantage of this rubber door when doingfluorescent microscopy because the ambient light would bring a lot ofnoise to the image sensor of the camera.

To capture good fluorescent microscopy image, it is desirable thatnearly no excitation light goes into the camera and only the fluorescentemitted by the sample is collected by the camera. For all commonsmartphones, however, the optical filter putting in front of the cameracannot block the undesired wavelength range of the light emitted fromthe light source of a smartphone very well due to the large divergenceangle of the beams emitted by the light source and the optical filternot working well for un-collimated beams. Collimation optics can bedesigned to collimated the beam emitted by the smartphone light sourceto address this issue, but this approach increase the size and cost ofthe adaptor. Instead, in this present invention, fluorescentillumination optics enables the excitation light to illuminate thesample partially from the waveguide inside the sample slide andpartially from the backside of the sample side in large obliqueincidence angle so that excitation light will nearly not be collected bythe camera to reduce the noise signal getting into the camera.

The bright-field illumination optics in the adaptor receive and turn thebeam emitted by the light source so as to back-illuminated the sample innormal incidence angle.

Typically, the optical box also comprises a lens mounted in it alignedwith the camera of the smartphone, which magnifies the images capturedby the camera. The images captured by the camera can be furtherprocessed by the processor of smartphone and outputs the analysis resulton the screen of smartphone.

To achieve both bright-field illumination and fluorescent illuminationoptics in a same optical adaptor, in this present invention, a slidablelever is used. The optical elements of the fluorescent illuminationoptics are mounted on the lever and when the lever fully slides into theoptical box, the fluorescent illumination optics elements block theoptical path of bright-field illumination optics and switch theillumination optics to fluorescent illumination optics. And when thelever slides out, the fluorescent illumination optics elements mountedon the lever move out of the optical path and switch the illuminationoptics to bright-field illumination optics. This lever design makes theoptical adaptor work in both bright-field and fluorescent illuminationmodes without the need for designing two different single-mode opticalboxes.

The lever comprises two planes at different planes at different heights.

In certain embodiments, two planes can be joined together with avertical bar and move together in or out of the optical box. In certainembodiments, two planes can be separated and each plane can moveindividually in or out of the optical box.

The upper lever plane comprises at least one optical element which canbe, but not limited to be an optical filter. The upper lever plane movesunder the light source and the preferred distance between the upperlever plane and the light source is in the range of 0 to 5 mm.

Part of the bottom lever plane is not parallel to the image plane. Andthe surface of the non-parallel part of the bottom lever plane hasmirror finish with high reflectivity larger than 95%. The non-parallelpart of the bottom lever plane moves under the light source and deflectsthe light emitted from the light source to back-illuminate the samplearea right under the camera. The preferred tilt angle of thenon-parallel part of the bottom lever plane is in the range of 45 degreeto 65 degree and the tilt angle is defined as the angle between thenon-parallel bottom plane and the vertical plane.

Part of the bottom lever plane is parallel to the image plane and islocated under and 1 mm to 10 mm away from the sample. The surface of theparallel part of the bottom lever plane is highly light absorptive withlight absorption larger than 95%. This absorptive surface is toeliminate the reflective light back-illuminating on the sample in smallincidence angle.

To slide in and out to switch the illumination optics using the lever, astopper design comprising a ball plunger and a groove on the lever isused in order to stop the lever at a pre-defined position when beingpulled outward from the adaptor. This allow the user to use arbitraryforce the pull the lever but make the lever to stop at a fixed positionwhere the optical adaptor's working mode is switched to bright-filedillumination.

A sample slider is mounted inside the receptacle slot to receive theQMAX device and position the sample in the QMAX device in the field ofview and focal range of the smartphone camera.

The sample slider comprises a fixed track frame and a moveable arm:

The frame track is fixedly mounted in the receptacle slot of the opticalbox. And the track frame has a sliding track slot that fits the widthand thickness of the QMAX device so that the QMAX device can slide alongthe track. The width and height of the track slot is carefullyconfigured to make the QMAX device shift less than 0.5 mm in thedirection perpendicular to the sliding direction in the sliding planeand shift less than less than 0.2 mm along the thickness direction ofthe QMAX device.

The frame track has an opened window under the field of view of thecamera of smartphone to allow the light back-illuminate the sample.

A moveable arm is pre-built in the sliding track slot of the track frameand moves together with the QMAX device to guide the movement of QMAXdevice in the track frame.

The moveable arm equipped with a stopping mechanism with two pre-definedstop positions. For one position, the arm will make the QMAX device stopat the position where a fixed sample area on the QMAX device is rightunder the camera of smartphone. For the other position, the arm willmake the QMAX device stop at the position where the sample area on QMAXdevice is out of the field of view of the smartphone and the QMAX devicecan be easily taken out of the track slot.

The moveable arm switches between the two stop positions by a pressingthe QMAX device and the moveable arm together to the end of the trackslot and then releasing.

The moveable arm can indicate if the QMAX device is inserted in correctdirection. The shape of one corner of the QMAX device is configured tobe different from the other three right angle corners. And the shape ofthe moveable arm matches the shape of the corner with the special shapeso that only in correct direction can QMAX device slide to correctposition in the track slot.

C) Smartphone/Detection System

Details of the Smartphone/Detection System are described in detail in avariety of publications including International Application (IA) No.PCT/US2016/046437 filed on Aug. 10, 2016, IA No. PCT/US2016/051775 filedSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application Nos. 62/456,287 and62/456,590, which were filed on Feb. 8, 2017, U.S. ProvisionalApplication No. 62/456,504, which was filed on Feb. 8, 2017, U.S.Provisional Application No. 62/459,544, which was filed on Feb. 15,2017, and U.S. Provisional Application Nos. 62/460,075 and 62/459,920,which were filed on Feb. 16, 2017, each of which are hereby incorporatedherein by reference in their entirety for all purposes.

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards for sample detection, analysis, and quantification. Incertain embodiments, the Q-card is used together with an adaptor thatcan connect the Q-card with a smartphone detection system. In certainembodiments, the smartphone comprises a camera and/or an illuminationsource. In certain embodiments, the smartphone comprises a camera, whichcan be used to capture images or the sample when the sample ispositioned in the field of view of the camera (e.g. by an adaptor). Incertain embodiments, the camera includes one set of lenses (e.g. as iniPhone™ 6). In certain embodiments, the camera includes at least twosets of lenses (e.g. as in iPhone™ 7). In certain embodiments, thesmartphone comprises a camera, but the camera is not used for imagecapturing.

In certain embodiments, the smartphone comprises a light source such asbut not limited to LED (light emitting diode). In certain embodiments,the light source is used to provide illumination to the sample when thesample is positioned in the field of view of the camera (e.g. by anadaptor). In certain embodiments, the light from the light source isenhanced, magnified, altered, and/or optimized by optical components ofthe adaptor.

In certain embodiments, the smartphone comprises a processor that isconfigured to process the information from the sample. The smartphoneincludes software instructions that, when executed by the processor, canenhance, magnify, and/or optimize the signals (e.g. images) from thesample. The processor can include one or more hardware components, suchas a central processing unit (CPU), an application-specific integratedcircuit (ASIC), an application-specific instruction-set processor(ASIP), a graphics processing unit (GPU), a physics processing unit(PPU), a digital signal processor (DSP), a field-programmable gate array(FPGA), a programmable logic device (PLD), a controller, amicrocontroller unit, a reduced instruction-set computer (RISC), amicroprocessor, or the like, or any combination thereof.

In certain embodiments, the smartphone comprises a communication unit,which is configured and/or used to transmit data and/or images relatedto the sample to another device. Merely by way of example, thecommunication unit can use a cable network, a wireline network, anoptical fiber network, a telecommunications network, an intranet, theInternet, a local area network (LAN), a wide area network (WAN), awireless local area network (WLAN), a metropolitan area network (MAN), awide area network (WAN), a public telephone switched network (PSTN), aBluetooth network, a ZigBee network, a near field communication (NFC)network, or the like, or any combination thereof. In certainembodiments, the smartphone is an iPhone™, an Android™ phone, or aWindows™ phone.

D) Method of Manufacture

Details of the Method of Manufacture are described in detail in avariety of publications including International Application No.PCT/US2018/057873 filed Oct. 26, 2018, which is hereby incorporated byreference herein for all purposes.

Devices of the disclosure can be fabricated using techniques well knownin the art. The choice of fabrication technique will depend on thematerial used for the device and the size of the spacer array and/or thesize of the spacers. Exemplary materials for fabricating the devices ofthe invention include glass, silicon, steel, nickel, polymers, e.g.,poly(methylmethacrylate) (PMMA), polycarbonate, polystyrene,polyethylene, polyolefins, silicones (e.g., poly(dimethylsiloxane)),polypropylene, cis-polyisoprene (rubber), poly(vinyl chloride) (PVC),poly(vinyl acetate) (PVAc), polychloroprene (neoprene),polytetrafluoroethylene (Teflon), poly(vinylidene chloride) (SaranA),and cyclic olefin polymer (COP) and cyclic olefin copolymer (COC), andcombinations thereof. Other materials are known in the art. For example,deep Reactive Ion Etch (DRIE) is used to fabricate silicon-based deviceswith small gaps, small spacers and large aspect ratios (ratio of spacerheight to lateral dimension). Thermoforming (embossing, injectionmolding) of plastic devices can also be used, e.g., when the smallestlateral feature is >20 microns and the aspect ratio of these features is10.

Additional methods include photolithography (e.g., stereolithography orx-ray photolithography), molding, embossing, silicon micromachining, wetor dry chemical etching, milling, diamond cutting, LithographieGalvanoformung and Abformung (LIGA), and electroplating. For example,for glass, traditional silicon fabrication techniques ofphotolithography followed by wet (KOH) or dry etching (reactive ionetching with fluorine or other reactive gas) can be employed. Techniquessuch as laser micromachining can be adopted for plastic materials withhigh photon absorption efficiency. This technique is suitable for lowerthroughput fabrication because of the serial nature of the process. Formass-produced plastic devices, thermoplastic injection molding, andcompression molding can be suitable. Conventional thermoplasticinjection molding used for mass-fabrication of compact discs (whichpreserves fidelity of features in sub-microns) can also be employed tofabricate the devices of the invention. For example, the device featuresare replicated on a glass master by conventional photolithography. Theglass master is electroformed to yield a tough, thermal shock resistant,thermally conductive, hard mold. This mold serves as the master templatefor injection molding or compression molding the features into a plasticdevice. Depending on the plastic material used to fabricate the devicesand the requirements on optical quality and throughput of the finishedproduct, compression molding or injection molding can be chosen as themethod of manufacture. Compression molding (also called hot embossing orrelief imprinting) has the advantages of being compatible with highmolecular weight polymers, which are excellent for small structures andcan replicate high aspect ratio structures but has longer cycle times.Injection molding works well for low aspect ratio structures and is mostsuitable for low molecular weight polymers.

A device can be fabricated in one or more pieces that are thenassembled. Layers of a device can be bonded together by clamps,adhesives, heat, anodic bonding, or reactions between surface groups(e.g., wafer bonding). Alternatively, a device with channels or gaps inmore than one plane can be fabricated as a single piece, e.g., usingstereolithography or other three-dimensional fabrication techniques.

To reduce non-specific adsorption of cells or compounds released bylysed cells onto the surfaces of the device, one or more surfaces of thedevice can be chemically modified to be non-adherent or repulsive. Thesurfaces can be coated with a thin film coating (e.g., a monolayer) ofcommercial non-stick reagents, such as those used to form hydrogels.Additional examples chemical species that can be used to modify thesurfaces of the device include oligoethylene glycols, fluorinatedpolymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid,bovine serum albumin, poly-vinyl alcohol, mucin, poly-HEMA,methacrylated PEG, and agarose. Charged polymers can also be employed torepel oppositely charged species. The type of chemical species used forrepulsion and the method of attachment to the surfaces of the devicewill depend on the nature of the species being repelled and the natureof the surfaces and the species being attached. Such surfacemodification techniques are well known in the art. The surfaces can befunctionalized before or after the device is assembled. The surfaces ofthe device can also be coated in order to capture materials in thesample, e.g., membrane fragments or proteins.

In certain embodiments of the present disclosure, a method forfabricating any Q-Card of the present disclosure can comprise injectionmolding of the first plate. In certain embodiments of the presentdisclosure, a method for fabricating any Q-Card of the presentdisclosure can comprise nanoimprinting or extrusion printing of thesecond plate. In certain embodiments of the present disclosure, a methodfor fabricating any Q-Card of the present disclosure can comprise Lasercutting the first plate. In certain embodiments of the presentdisclosure, a method for fabricating any Q-Card of the presentdisclosure can comprise nanoimprinting or extrusion printing of thesecond plate. In certain embodiments of the present disclosure, a methodfor fabricating any Q-Card of the present disclosure can compriseinjection molding and laser cutting the first plate. In certainembodiments of the present disclosure, a method for fabricating anyQ-Card of the present disclosure can comprise nanoimprinting orextrusion printing of the second plate. In certain embodiments of thepresent disclosure, a method for fabricating any Q-Card of the presentdisclosure can comprise nanoimprinting or extrusion printing tofabricated both the first and the second plate. In certain embodimentsof the present disclosure, a method for fabricating any Q-Card of thepresent disclosure can comprise fabricating the first plate or thesecond plate, using injection molding, laser cutting the first plate,nanoimprinting, extrusion printing, or a combination of thereof. Incertain embodiments of the present disclosure, a method for fabricatingany Q-Card of the present disclosure can comprise a step of attachingthe hinge on the first and the second plates after the fabrication ofthe first and second plates.

E) Sample Types & Subjects

Details of the Samples & Subjects are described in detail in a varietyof publications including International Application (IA) No.PCT/US2016/046437 filed on Aug. 10, 2016, IA No. PCT/US2016/051775 filedon Sep. 14, 2016, IA No. PCT/US201/017307 filed on Feb. 7, 2018, IA No.and PCT/US2017/065440 filed on Dec. 8, 2017, each of which is herebyincorporated by reference herein for all purposes.

A sample can be obtained from a subject. A subject as described hereincan be of any age and can be an adult, infant or child. In some cases,the subject is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 years old, or within arange therein (e.g., between 2 and 20 years old, between 20 and 40 yearsold, or between 40 and 90 years old). A particular class of subjectsthat can benefit is subjects who have or are suspected of having aninfection (e.g., a bacterial and/or a viral infection). Anotherparticular class of subjects that can benefit is subjects who can be athigher risk of getting an infection. Furthermore, a subject treated byany of the methods or compositions described herein can be male orfemale. Any of the methods, devices, or kits disclosed herein can alsobe performed on a non-human subject, such as a laboratory or farmanimal. Non-limiting examples of a non-human subjects include a dog, agoat, a guinea pig, a hamster, a mouse, a pig, a non-human primate(e.g., a gorilla, an ape, an orangutan, a lemur, or a baboon), a rat, asheep, a cow, or a zebrafish.

The devices, apparatus, systems, and methods herein disclosed can beused for samples such as but not limited to diagnostic samples, clinicalsamples, environmental samples and foodstuff samples.

For example, in certain embodiments, the devices, apparatus, systems,and methods herein disclosed are used for a sample that includes cells,tissues, bodily fluids and/or a mixture thereof. In certain embodiments,the sample comprises a human body fluid. In certain embodiments, thesample comprises at least one of cells, tissues, bodily fluids, stool,amniotic fluid, aqueous humour, vitreous humour, blood, whole blood,fractionated blood, plasma, serum, breast milk, cerebrospinal fluid,cerumen, chyle, chime, endolymph, perilymph, feces, gastric acid,gastric juice, lymph, mucus, nasal drainage, phlegm, pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen,sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled breathcondensate.

In certain embodiments, the devices, apparatus, systems, and methodsherein disclosed are used for an environmental sample that is obtainedfrom any suitable source, such as but not limited to: river, lake, pond,ocean, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water,drinking water, etc.; solid samples from soil, compost, sand, rocks,concrete, wood, brick, sewage, etc.; and gaseous samples from the air,underwater heat vents, industrial exhaust, vehicular exhaust, etc. Incertain embodiments, the environmental sample is fresh from the source;in certain embodiments, the environmental sample is processed. Forexample, samples that are not in liquid form are converted to liquidform before the subject devices, apparatus, systems, and methods areapplied.

In certain embodiments, the devices, apparatus, systems, and methodsherein disclosed are used for a foodstuff sample, which is suitable orhas the potential to become suitable for animal consumption, e.g., humanconsumption. In certain embodiments, a foodstuff sample includes rawingredients, cooked or processed food, plant and animal sources of food,preprocessed food as well as partially or fully processed food, etc. Incertain embodiments, samples that are not in liquid form are convertedto liquid form before the subject devices, apparatus, systems, andmethods are applied.

The subject devices, apparatus, systems, and methods can be used toanalyze any volume of the sample. Examples of the volumes include, butare not limited to, about 10 mL or less, 5 mL or less, 3 mL or less, 1microliter (uL, also “uL” herein) or less, 500 uL or less, 300 uL orless, 250 uL or less, 200 uL or less, 170 uL or less, 150 uL or less,125 uL or less, 100 uL or less, 75 uL or less, 50 uL or less, 25 uL orless, 20 uL or less, 15 uL or less, 10 uL or less, 5 uL or less, 3 uL orless, 1 uL or less, 0.5 uL or less, 0.1 uL or less, 0.05 uL or less,0.001 uL or less, 0.0005 uL or less, 0.0001 uL or less, 10 pL or less, 1pL or less, or a range between any two of the values.

In certain embodiments, the volume of the sample includes, but is notlimited to, about 100 uL or less, 75 uL or less, 50 uL or less, 25 uL orless, 20 uL or less, 15 uL or less, 10 uL or less, 5 uL or less, 3 uL orless, 1 uL or less, 0.5 uL or less, 0.1 uL or less, 0.05 uL or less,0.001 uL or less, 0.0005 uL or less, 0.0001 uL or less, 10 pL or less, 1pL or less, or a range between any two of the values. In certainembodiments, the volume of the sample includes, but is not limited to,about 10 uL or less, 5 uL or less, 3 uL or less, 1 uL or less, 0.5 uL orless, 0.1 uL or less, 0.05 uL or less, 0.001 uL or less, 0.0005 uL orless, 0.0001 uL or less, 10 pL or less, 1 pL or less, or a range betweenany two of the values.

In certain embodiments, the amount of the sample is about a drop ofliquid. In certain embodiments, the amount of sample is the amountcollected from a pricked finger or fingerstick. In certain embodiments,the amount of sample is the amount collected from a microneedle,micropipette or a venous draw.

F) Machine Learning

Details of the Network are described in detail in a variety ofpublications including International Application (IA) No.PCT/US2018/017504 filed Feb. 8, 2018, and PCT/US2018/057877 filed Oct.26, 2018, each of which are hereby incorporated by reference herein forall purposes.

One aspect of the present invention provides a framework of machinelearning and deep learning for analyte detection and localization. Amachine learning algorithm is an algorithm that is able to learn fromdata. A more rigorous definition of machine learning is “A computerprogram is said to learn from experience E with respect to some class oftasks T and performance measure P, if its performance at tasks in T, asmeasured by P, improves with experience E.” It explores the study andconstruction of algorithms that can learn from and make predictions ondata—such algorithms overcome the static program instructions by makingdata driven predictions or decisions, through building a model fromsample inputs.

Deep learning is a specific kind of machine learning based on a set ofalgorithms that attempt to model high level abstractions in data. In asimple case, there might be two sets of neurons: ones that receive aninput signal and ones that send an output signal. When the input layerreceives an input, it passes on a modified version of the input to thenext layer. In a deep network, there are many layers between the inputand output (and the layers are not made of neurons but it can help tothink of it that way), allowing the algorithm to use multiple processinglayers, composed of multiple linear and non-linear transformations.

One aspect of the present invention is to provide two analyte detectionand localization approaches. The first approach is a deep learningapproach and the second approach is a combination of deep learning andcomputer vision approaches.

(i) Deep Learning Approach.

In the first approach, the disclosed analyte detection and localizationworkflow consists of two stages, training and prediction. We describetraining and prediction stages in the following paragraphs.

(a) Training Stage

In the training stage, training data with annotation is fed into aconvolutional neural network. Convolutional neural network is aspecialized neural network for processing data that has a grid-like,feed forward and layered network topology. Examples of the data includetime-series data, which can be thought of as a 1D grid taking samples atregular time intervals, and image data, which can be thought of as a 2Dgrid of pixels. Convolutional networks have been successful in practicalapplications. The name “convolutional neural network” indicates that thenetwork employs a mathematical operation called convolution. Convolutionis a specialized kind of linear operation. Convolutional networks aresimply neural networks that use convolution in place of general matrixmultiplication in at least one of their layers.

The machine learning model receives one or multiple images of samplesthat contain the analytes taken by the imager over the sample holdingQMAX device as training data. Training data are annotated for analytesto be assayed, wherein the annotations indicate whether or not analytesare in the training data and where they locate in the image. Annotationcan be done in the form of tight bounding boxes which fully contains theanalyte, or center locations of analytes. In the latter case, centerlocations are further converted into circles covering analytes or aGaussian kernel in a point map.

When the size of training data is large, training machine learning modelpresents two challenges: annotation (usually done by human) is timeconsuming, and the training is computationally expensive. To overcomethese challenges, one can partition the training data into patches ofsmall size, then annotate and train on these patches, or a portion ofthese patches. The term “machine learning” can refer to algorithms,systems and apparatus in the field of artificial intelligence that oftenuse statistical techniques and artificial neural network trained fromdata without being explicitly programmed.

The annotated images are fed to the machine learning (ML) trainingmodule, and the model trainer in the machine learning module will traina ML model from the training data (annotated sample images). The inputdata will be fed to the model trainer in multiple iterations untilcertain stopping criterion is satisfied. The output of the ML trainingmodule is a ML model—a computational model that is built from a trainingprocess in the machine learning from the data that gives computer thecapability to perform certain tasks (e.g. detect and classify theobjects) on its own.

The trained machine learning model is applied during the predication (orinference) stage by the computer. Examples of machine learning modelsinclude ResNet, DenseNet, etc. which are also named as “deep learningmodels” because of the depth of the connected layers in their networkstructure. In certain embodiments, the Caffe library with fullyconvolutional network (FCN) was used for model training and predication,and other convolutional neural network architecture and library can alsobe used, such as TensorFlow.

The training stage generates a model that will be used in the predictionstage. The model can be repeatedly used in the prediction stage forassaying the input. Thus, the computing unit only needs access to thegenerated model. It does not need access to the training data, norrequiring the training stage to be run again on the computing unit.

(b) Prediction Stage

In the predication/inference stage, a detection component is applied tothe input image, and an input image is fed into the predication(inference) module preloaded with a trained model generated from thetraining stage. The output of the prediction stage can be bounding boxesthat contain the detected analytes with their center locations or apoint map indicating the location of each analyte, or a heatmap thatcontains the information of the detected analytes.

When the output of the prediction stage is a list of bounding boxes, thenumber of analytes in the image of the sample for assaying ischaracterized by the number of detected bounding boxes. When the outputof the prediction stage is a point map, the number of analytes in theimage of the sample for assaying is characterized by the integration ofthe point map. When the output of the prediction is a heatmap, alocalization component is used to identify the location and the numberof detected analytes is characterized by the entries of the heatmap.

One embodiment of the localization algorithm is to sort the heatmapvalues into a one-dimensional ordered list, from the highest value tothe lowest value. Then pick the pixel with the highest value, remove thepixel from the list, along with its neighbors. Iterate the process topick the pixel with the highest value in the list, until all pixels areremoved from the list. In the detection component using heatmap, aninput image, along with the model generated from the training stage, isfed into a convolutional neural network, and the output of the detectionstage is a pixel-level prediction, in the form of a heatmap. The heatmapcan have the same size as the input image, or it can be a scaled downversion of the input image, and it is the input to the localizationcomponent. We disclose an algorithm to localize the analyte center. Themain idea is to iteratively detect local peaks from the heatmap. Afterthe peak is localized, we calculate the local area surrounding the peakbut with smaller value. We remove this region from the heatmap and findthe next peak from the remaining pixels. The process is repeated onlyall pixels are removed from the heatmap.

In certain embodiments, the present invention provides the localizationalgorithm to sort the heatmap values into a one-dimensional orderedlist, from the highest value to the lowest value. Then pick the pixelwith the highest value, remove the pixel from the list, along with itsneighbors. Iterate the process to pick the pixel with the highest valuein the list, until all pixels are removed from the list.

Algorithm GlobalSearch (heatmap) Input: heatmap Output: loci loci ←{ }sort(heatmap) while (heatmap is not empty) { s ← pop(heatmap) D ← {diskcenter as s with radius R} heatmap = heatmap \ D // remove D from theheatmap add s to loci }

After sorting, heatmap is a one-dimensional ordered list, where theheatmap value is ordered from the highest to the lowest. Each heatmapvalue is associated with its corresponding pixel coordinates. The firstitem in the heatmap is the one with the highest value, which is theoutput of the pop(heatmap) function. One disk is created, where thecenter is the pixel coordinate of the one with highest heatmap value.Then all heatmap values whose pixel coordinates resides inside the diskis removed from the heatmap. The algorithm repeatedly pops up thehighest value in the current heatmap, removes the disk around it, tillthe items are removed from the heatmap.

In the ordered list heatmap, each item has the knowledge of theproceeding item, and the following item. When removing an item from theordered list, we make the following changes:

-   -   Assume the removing item is x₁, its proceeding item is x_(p),        and its following item is x_(f).    -   For the proceeding item x_(p), re-define its following item to        the following item of the removing item. Thus, the following        item of x_(p) is now x_(f).    -   For the removing item x_(r), un-define its proceeding item and        following item, which removes it from the ordered list.    -   For the following item x_(f), re-define its proceeding item to        the proceeding item of the removed item. Thus, the proceeding        item of x_(f) is now x_(p).

After all items are removed from the ordered list, the localizationalgorithm is complete. The number of elements in the set loci will bethe count of analytes, and location information is the pixel coordinatefor each s in the set loci.

Another embodiment searches local peak, which is not necessary the onewith the highest heatmap value. To detect each local peak, we start froma random starting point, and search for the local maximal value. Afterwe find the peak, we calculate the local area surrounding the peak butwith smaller value. We remove this region from the heatmap and find thenext peak from the remaining pixels. The process is repeated only allpixels are removed from the heatmap.

Algorithm LocalSearch (s, heatmap) Input: s: starting location (x, y)heatmap Output: s: location of local peak. We only consider pixels ofvalue > 0. Algorithm Cover (s, heatmap) Input: s: location of localpeak. heatmap: Output: cover: a set of pixels covered by peak:

This is a breadth-first-search algorithm starting from s, with onealtered condition of visiting points: a neighbor p of the currentlocation q is only added to cover if heatmap[p]>0 andheatmap[p]<=heatmap[q]. Therefore, each pixel in cover has anon-descending path leading to the local peak s.

Algorithm Localization (heatmap) Input: heatmap Output: loci loci ←{ }pixels ←{all pixels from heatmap} while pixels is not empty { s ←anypixel from pixels s ←LocalSearch(s, heatmap)  // s is now local peakprobe local region of radius R surrounding s for better local peak r←Cover(s, heatmap) pixels ← pixels \ r // remove all pixels in cover adds to loci

(ii) Mixture of Deep Learning and Computer Vision Approaches.

In the second approach, the detection and localization are realized bycomputer vision algorithms, and a classification is realized by deeplearning algorithms, wherein the computer vision algorithms detect andlocate possible candidates of analytes, and the deep learning algorithmclassifies each possible candidate as a true analyte and false analyte.The location of all true analyte (along with the total count of trueanalytes) will be recorded as the output.

(a) Detection.

The computer vision algorithm detects possible candidate based on thecharacteristics of analytes, including but not limited to intensity,color, size, shape, distribution, etc. A pre-processing scheme canimprove the detection. Pre-processing schemes include contrastenhancement, histogram adjustment, color enhancement, de-nosing,smoothing, de-focus, etc. After pre-processing, the input image is sentto a detector. The detector tells the existing of possible candidate ofanalyte and gives an estimate of its location. The detection can bebased on the analyte structure (such as edge detection, line detection,circle detection, etc.), the connectivity (such as blob detection,connect components, contour detection, etc.), intensity, color, shapeusing schemes such as adaptive thresholding, etc.

(b) Localization.

After detection, the computer vision algorithm locates each possiblecandidate of analytes by providing its boundary or a tight bounding boxcontaining it. This can be achieved through object segmentationalgorithms, such as adaptive thresholding, background subtraction,floodfill, mean shift, watershed, etc. Very often, the localization canbe combined with detection to produce the detection results along withthe location of each possible candidates of analytes.

(c) Classification.

The deep learning algorithms, such as convolutional neural networks,achieve start-of-the-art visual classification. We employ deep learningalgorithms for classification on each possible candidate of analytes.Various convolutional neural network can be utilized for analyteclassification, such as VGGNet, ResNet, MobileNet, DenseNet, etc.

Given each possible candidate of analyte, the deep learning algorithmcomputes through layers of neurons via convolution filters andnon-linear filters to extract high-level features that differentiateanalyte against non-analytes. A layer of fully convolutional networkwill combine high-level features into classification results, whichtells whether it is a true analyte or not, or the probability of being aanalyte.

G) Applications, Bio/Chemical Biomarkers, and Health Conditions

The applications of the present invention include, but not limited to,(a) the detection, purification and quantification of chemical compoundsor biomolecules that correlates with the stage of certain diseases,e.g., infectious and parasitic disease, injuries, cardiovasculardisease, cancer, mental disorders, neuropsychiatric disorders andorganic diseases, e.g., pulmonary diseases, renal diseases, (b) thedetection, purification and quantification of microorganism, e.g.,virus, fungus and bacteria from environment, e.g., water, soil, orbiological samples, e.g., tissues, bodily fluids, (c) the detection,quantification of chemical compounds or biological samples that posehazard to food safety or national security, e.g. toxic waste, anthrax,(d) quantification of vital parameters in medical or physiologicalmonitor, e.g., glucose, blood oxygen level, total blood count, (e) thedetection and quantification of specific DNA or RNA from biosamples,e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing ofgenetic sequences in DNA in the chromosomes and mitochondria for genomeanalysis or (g) to detect reaction products, e.g., during synthesis orpurification of pharmaceuticals.

The detection can be carried out in various sample matrix, such ascells, tissues, bodily fluids, and stool. Bodily fluids of interestinclude but are not limited to, amniotic fluid, aqueous humour, vitreoushumour, blood (e.g., whole blood, fractionated blood, plasma, serum,etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus (including nasal drainage and phlegm), pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil),semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaledcondensate. In certain embodiments, the sample comprises a human bodyfluid. In certain embodiments, the sample comprises at least one ofcells, tissues, bodily fluids, stool, amniotic fluid, aqueous humour,vitreous humour, blood, whole blood, fractionated blood, plasma, serum,breast milk, cerebrospinal fluid, cerumen, chyle, chime, endolymph,perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasaldrainage, phlegm, pericardial fluid, peritoneal fluid, pleural fluid,pus, rheum, saliva, sebum, semen, sputum, sweat, synovial fluid, tears,vomit, urine, and exhaled condensate.

In embodiments, the sample is at least one of a biological sample, anenvironmental sample, and a biochemical sample.

The devices, systems and the methods in the present invention find usein a variety of different applications in various fields, wheredetermination of the presence or absence, and/or quantification of oneor more analytes in a sample are desired. For example, the subjectmethod finds use in the detection of proteins, peptides, nucleic acids,synthetic compounds, inorganic compounds, and the like. The variousfields include, but not limited to, human, veterinary, agriculture,foods, environments, drug testing, and others.

In certain embodiments, the subject method finds use in the detection ofnucleic acids, proteins, or other biomolecules in a sample. The methodscan include the detection of a set of biomarkers, e.g., two or moredistinct protein or nucleic acid biomarkers, in a sample. For example,the methods can be used in the rapid, clinical detection of two or moredisease biomarkers in a biological sample, e.g., as can be employed inthe diagnosis of a disease condition in a subject, or in the ongoingmanagement or treatment of a disease condition in a subject, etc. Asdescribed above, communication to a physician or other health-careprovider can better ensure that the physician or other health-careprovider is made aware of, and cognizant of, possible concerns and canthus be more likely to take appropriate action.

The applications of the devices, systems and methods in the presentinventions of employing a CROF device include, but are not limited to,(a) the detection, purification and quantification of chemical compoundsor biomolecules that correlates with the stage of certain diseases,e.g., infectious and parasitic disease, injuries, cardiovasculardisease, cancer, mental disorders, neuropsychiatric disorders andorganic diseases, e.g., pulmonary diseases, renal diseases, (b) thedetection, purification and quantification of microorganism, e.g.,virus, fungus and bacteria from environment, e.g., water, soil, orbiological samples, e.g., tissues, bodily fluids, (c) the detection,quantification of chemical compounds or biological samples that posehazard to food safety or national security, e.g. toxic waste, anthrax,(d) quantification of vital parameters in medical or physiologicalmonitor, e.g., glucose, blood oxygen level, total blood count, (e) thedetection and quantification of specific DNA or RNA from biosamples,e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing ofgenetic sequences in DNA in the chromosomes and mitochondria for genomeanalysis or (g) to detect reaction products, e.g., during synthesis orpurification of pharmaceuticals. Some of the specific applications ofthe devices, systems and methods in the present invention are describednow in further detail.

The applications of the present invention include, but not limited to,(a) the detection, purification and quantification of chemical compoundsor biomolecules that correlates with the stage of certain diseases,e.g., infectious and parasitic disease, injuries, cardiovasculardisease, cancer, mental disorders, neuropsychiatric disorders andorganic diseases, e.g., pulmonary diseases, renal diseases, (b) thedetection, purification and quantification of microorganism, e.g.,virus, fungus and bacteria from environment, e.g., water, soil, orbiological samples, e.g., tissues, bodily fluids, (c) the detection,quantification of chemical compounds or biological samples that posehazard to food safety or national security, e.g. toxic waste, anthrax,(d) quantification of vital parameters in medical or physiologicalmonitor, e.g., glucose, blood oxygen level, total blood count, (e) thedetection and quantification of specific DNA or RNA from biosamples,e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing ofgenetic sequences in DNA in the chromosomes and mitochondria for genomeanalysis or (g) to detect reaction products, e.g., during synthesis orpurification of pharmaceuticals.

An implementation of the devices, systems and methods in the presentinvention can include a) obtaining a sample, b) applying the sample toCROF device containing a capture agent that binds to an analyte ofinterest, under conditions suitable for binding of the analyte in asample to the capture agent, c) washing the CROF device, and d) readingthe CROF device, thereby obtaining a measurement of the amount of theanalyte in the sample. In certain embodiments, the analyte can be abiomarker, an environmental marker, or a foodstuff marker. The sample insome instances is a liquid sample, and can be a diagnostic sample (suchas saliva, serum, blood, sputum, urine, sweat, lacrima, semen, ormucus); an environmental sample obtained from a river, ocean, lake,rain, snow, sewage, sewage processing runoff, agricultural runoff,industrial runoff, tap water or drinking water; or a foodstuff sampleobtained from tap water, drinking water, prepared food, processed foodor raw food.

In any embodiment, the CROF device can be placed in a microfluidicdevice and the applying step b) can include applying a sample to amicrofluidic device comprising the CROF device.

In any embodiment, the reading step d) can include detecting afluorescence or luminescence signal from the CROF device.

In any embodiment, the reading step d) can include reading the CROFdevice with a handheld device configured to read the CROF device. Thehandheld device can be a mobile phone, e.g., a smart phone.

In any embodiment, the CROF device can include a labeling agent that canbind to an analyte-capture agent complex on the CROF device.

In any embodiment, the devices, systems and methods in the presentinvention can further include, between steps c) and d), the steps ofapplying to the CROF device a labeling agent that binds to ananalyte-capture agent complex on the CROF device, and washing the CROFdevice.

In any embodiment, the reading step d) can include reading an identifierfor the CROF device. The identifier can be an optical barcode, a radiofrequency ID tag, or combinations thereof.

In any embodiment, the devices, systems and methods in the presentinvention can further include applying a control sample to a controlCROF device containing a capture agent that binds to the analyte,wherein the control sample includes a known detectable amount of theanalyte, and reading the control CROF device, thereby obtaining acontrol measurement for the known detectable amount of the analyte in asample.

In any embodiment, the sample can be a diagnostic sample obtained from asubject, the analyte can be a biomarker, and the measured amount of theanalyte in the sample can be diagnostic of a disease or a condition.

In any embodiment, the devices, systems and methods in the presentinvention can further include receiving or providing to the subject areport that indicates the measured amount of the biomarker and a rangeof measured values for the biomarker in an individual free of or at lowrisk of having the disease or condition, wherein the measured amount ofthe biomarker relative to the range of measured values is diagnostic ofa disease or condition.

In any embodiment, the devices, systems and methods in the presentinvention can further include diagnosing the subject based oninformation including the measured amount of the biomarker in thesample. In some cases, the diagnosing step includes sending datacontaining the measured amount of the biomarker to a remote location andreceiving a diagnosis based on information including the measurementfrom the remote location.

In any embodiment, the applying step b) can include isolating miRNA fromthe sample to generate an isolated miRNA sample, and applying theisolated miRNA sample to the disk-coupled dots-on-pillar antenna (CROFdevice) array.

In any embodiment, the method can include receiving or providing areport that indicates the safety or harmfulness for a subject to beexposed to the environment from which the sample was obtained.

In any embodiment, the method can include sending data containing themeasured amount of the environmental marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to be exposed to the environment from which the sample wasobtained.

In any embodiment, the CROF device array can include a plurality ofcapture agents that each binds to an environmental marker, and whereinthe reading step d) can include obtaining a measure of the amount of theplurality of environmental markers in the sample. In any embodiment, thesample can be a foodstuff sample, wherein the analyte can be a foodstuffmarker, and wherein the amount of the foodstuff marker in the sample cancorrelate with safety of the foodstuff for consumption.

In any embodiment, the method can include receiving or providing areport that indicates the safety or harmfulness for a subject to consumethe foodstuff from which the sample is obtained.

In any embodiment, the method can include sending data containing themeasured amount of the foodstuff marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to consume the foodstuff from which the sample is obtained. Inany embodiment, the CROF device array can include a plurality of captureagents that each binds to a foodstuff marker, wherein the obtaining caninclude obtaining a measure of the amount of the plurality of foodstuffmarkers in the sample, and wherein the amount of the plurality offoodstuff marker in the sample can correlate with safety of thefoodstuff for consumption.

Also provided herein are kits that find use in practicing the devices,systems and methods in the present invention.

The amount of sample can be about a drop of a sample. The amount ofsample can be the amount collected from a pricked finger or fingerstick.The amount of sample can be the amount collected from a microneedle or avenous draw.

A sample can be used without further processing after obtaining it fromthe source, or can be processed, e.g., to enrich for an analyte ofinterest, remove large particulate matter, dissolve or resuspend a solidsample, etc.

Any suitable method of applying a sample to the CROF device can beemployed. Suitable methods can include using a pipet, dropper, syringe,etc. In certain embodiments, when the CROF device is located on asupport in a dipstick format, as described below, the sample can beapplied to the CROF device by dipping a sample-receiving area of thedipstick into the sample.

A sample can be collected at one time, or at a plurality of times.Samples collected over time can be aggregated and/or processed (byapplying to a CROF device and obtaining a measurement of the amount ofanalyte in the sample, as described herein) individually. In someinstances, measurements obtained over time can be aggregated and can beuseful for longitudinal analysis over time to facilitate screening,diagnosis, treatment, and/or disease prevention.

Washing the CROF device to remove unbound sample components can be donein any convenient manner, as described above. In certain embodiments,the surface of the CROF device is washed using binding buffer to removeunbound sample components. Detectable labeling of the analyte can bedone by any convenient method. The analyte can be labeled directly orindirectly. In direct labeling, the analyte in the sample is labeledbefore the sample is applied to the CROF device. In indirect labeling,an unlabeled analyte in a sample is labeled after the sample is appliedto the CROF device to capture the unlabeled analyte, as described below.

The samples from a subject, the health of a subject, and otherapplications of the present invention are further described below.Exemplary samples, health conditions, and application are also disclosedin, e.g., U.S. Pub. Nos. 2014/0154668 and 2014/0045209, which are herebyincorporated by reference.

The present inventions find use in a variety of applications, where suchapplications are generally analyte detection applications in which thepresence of a particular analyte in a given sample is detected at leastqualitatively, if not quantitatively. Protocols for carrying out analytedetection assays are well known to those of skill in the art and neednot be described in great detail here. Generally, the sample suspectedof comprising an analyte of interest is contacted with the surface of asubject nanosensor under conditions sufficient for the analyte to bindto its respective capture agent that is tethered to the sensor. Thecapture agent has highly specific affinity for the targeted molecules ofinterest. This affinity can be antigen-antibody reaction whereantibodies bind to specific epitope on the antigen, or a DNA/RNA orDNA/RNA hybridization reaction that is sequence-specific between two ormore complementary strands of nucleic acids. Thus, if the analyte ofinterest is present in the sample, it likely binds to the sensor at thesite of the capture agent and a complex is formed on the sensor surface.Namely, the captured analytes are immobilized at the sensor surface.After removing the unbounded analytes, the presence of this bindingcomplex on the surface of the sensor (e.g. the immobilized analytes ofinterest) is then detected, e.g., using a labeled secondary captureagent.

Specific analyte detection applications of interest includehybridization assays in which the nucleic acid capture agents areemployed and protein binding assays in which polypeptides, e.g.,antibodies, are employed. In these assays, a sample is first preparedand following sample preparation, the sample is contacted with a subjectnanosensor under specific binding conditions, whereby complexes areformed between target nucleic acids or polypeptides (or other molecules)that are complementary to capture agents attached to the sensor surface.

In one embodiment, the capture oligonucleotide is synthesized singlestrand DNA of 20-100 bases length, that is thiolated at one end. Thesemolecules are are immobilized on the nanodevices' surface to capture thetargeted single-strand DNA (which can be at least 50 bp length) that hasa sequence that is complementary to the immobilized capture DNA. Afterthe hybridization reaction, a detection single strand DNA (which can beof 20-100 bp in length) whose sequence are complementary to the targetedDNA's unoccupied nucleic acid is added to hybridize with the target. Thedetection DNA has its one end conjugated to a fluorescence label, whoseemission wavelength are within the plasmonic resonance of thenanodevice. Therefore by detecting the fluorescence emission emanatefrom the nanodevices' surface, the targeted single strand DNA can beaccurately detected and quantified. The length for capture and detectionDNA determine the melting temperature (nucleotide strands will separateabove melting temperature), the extent of misparing (the longer thestrand, the lower the misparing).

One of the concerns of choosing the length for complementary bindingdepends on the needs to minimize misparing while keeping the meltingtemperature as high as possible. In addition, the total length of thehybridization length is determined in order to achieve optimum signalamplification.

A subject sensor can be employed in a method of diagnosing a disease orcondition, comprising: (a) obtaining a liquid sample from a patientsuspected of having the disease or condition, (b) contacting the samplewith a subject nanosensor, wherein the capture agent of the nanosensorspecifically binds to a biomarker for the disease and wherein thecontacting is done under conditions suitable for specific binding of thebiomarker with the capture agent; (c) removing any biomarker that is notbound to the capture agent; and (d) reading a light signal frombiomarker that remain bound to the nanosensor, wherein a light signalindicates that the patient has the disease or condition, wherein themethod further comprises labeling the biomarker with a light-emittinglabel, either prior to or after it is bound to the capture agent. Aswill be described in greater detail below, the patient can suspected ofhaving cancer and the antibody binds to a cancer biomarker. In otherembodiments, the patient is suspected of having a neurological disorderand the antibody binds to a biomarker for the neurological disorder.

The applications of the subject sensor include, but not limited to, (a)the detection, purification and quantification of chemical compounds orbiomolecules that correlates with the stage of certain diseases, e.g.,infectious and parasitic disease, injuries, cardiovascular disease,cancer, mental disorders, neuropsychiatric disorders and organicdiseases, e.g., pulmonary diseases, renal diseases, (b) the detection,purification and quantification of microorganism, e.g., virus, fungusand bacteria from environment, e.g., water, soil, or biological samples,e.g., tissues, bodily fluids, (c) the detection, quantification ofchemical compounds or biological samples that pose hazard to food safetyor national security, e.g. toxic waste, anthrax, (d) quantification ofvital parameters in medical or physiological monitor, e.g., glucose,blood oxygen level, total blood count, (e) the detection andquantification of specific DNA or RNA from biosamples, e.g., cells,viruses, bodily fluids, (f) the sequencing and comparing of geneticsequences in DNA in the chromosomes and mitochondria for genome analysisor (g) to detect reaction products, e.g., during synthesis orpurification of pharmaceuticals.

The detection can be carried out in various sample matrix, such ascells, tissues, bodily fluids, and stool. Bodily fluids of interestinclude but are not limited to, amniotic fluid, aqueous humour, vitreoushumour, blood (e.g., whole blood, fractionated blood, plasma, serum,etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus (including nasal drainage and phlegm), pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil),semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaledcondensate.

In certain embodiments, a subject biosensor can be used diagnose apathogen infection by detecting a target nucleic acid from a pathogen ina sample. The target nucleic acid can be, for example, from a virus thatis selected from the group comprising human immunodeficiency virus 1 and2 (HIV-1 and HIV-2), human T-cell leukaemia virus and 2 (HTLV-1 andHTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis Bvirus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), humanpapillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes-simplex virus 1 and 2 (HSV-1 and HSV-2), human herpesvirus8 (HHV-8, also known as Kaposi sarcoma herpesvirus) and flaviviruses,including yellow fever virus, dengue virus, Japanese encephalitis virus,West Nile virus and Ebola virus. The present invention is not, however,limited to the detection of nucleic acid, e.g., DNA or RNA, sequencesfrom the aforementioned viruses, but can be applied without any problemto other pathogens important in veterinary and/or human medicine.

Human papillomaviruses (HPV) are further subdivided on the basis oftheir DNA sequence homology into more than 70 different types. Thesetypes cause different diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17 and 19-25 and46-50 cause lesions in patients with a weakened immune system. Types 6,11, 34, 39, 41-44 and 51-55 cause benign acuminate warts on the mucosaeof the genital region and of the respiratory tract. HPV types 16 and 18are of special medical interest, as they cause epithelial dysplasias ofthe genital mucosa and are associated with a high proportion of theinvasive carcinomas of the cervix, vagina, vulva and anal canal.Integration of the DNA of the human papillomavirus is considered to bedecisive in the carcinogenesis of cervical cancer. Humanpapillomaviruses can be detected for example from the DNA sequence oftheir capsid proteins L1 and L2. Accordingly, the method of the presentinvention is especially suitable for the detection of DNA sequences ofHPV types 16 and/or 18 in tissue samples, for assessing the risk ofdevelopment of carcinoma.

In some cases, the nanosensor can be employed to detect a biomarker thatis present at a low concentration. For example, the nanosensor can beused to detect cancer antigens in a readily accessible bodily fluids(e.g., blood, saliva, urine, tears, etc.), to detect biomarkers fortissue-specific diseases in a readily accessible bodily fluid (e.g., abiomarkers for a neurological disorder (e.g., Alzheimer's antigens)), todetect infections (particularly detection of low titer latent viruses,e.g., HIV), to detect fetal antigens in maternal blood, and fordetection of exogenous compounds (e.g., drugs or pollutants) in asubject's bloodstream, for example.

The following table provides a list of protein biomarkers that can bedetected using the subject nanosensor (when used in conjunction with anappropriate monoclonal antibody), and their associated diseases. Onepotential source of the biomarker (e.g., “CSF”; cerebrospinal fluid) isalso indicated in the table. In many cases, the subject biosensor candetect those biomarkers in a different bodily fluid to that indicated.For example, biomarkers that are found in CSF can be identified inurine, blood or saliva.

H) Utility

The subject method finds use in a variety of different applicationswhere determination of the presence or absence, and/or quantification ofone or more analytes in a sample are desired. For example, the subjectmethod finds use in the detection of proteins, peptides, nucleic acids,synthetic compounds, inorganic compounds, and the like.

In certain embodiments, the subject method finds use in the detection ofnucleic acids, proteins, or other biomolecules in a sample. The methodscan include the detection of a set of biomarkers, e.g., two or moredistinct protein or nucleic acid biomarkers, in a sample. For example,the methods can be used in the rapid, clinical detection of two or moredisease biomarkers in a biological sample, e.g., as can be employed inthe diagnosis of a disease condition in a subject, or in the ongoingmanagement or treatment of a disease condition in a subject, etc. Asdescribed above, communication to a physician or other health-careprovider can better ensure that the physician or other health-careprovider is made aware of, and cognizant of, possible concerns and canthus be more likely to take appropriate action.

The applications of the devices, systems and methods in the presentinvention of employing a CROF device include, but are not limited to,(a) the detection, purification and quantification of chemical compoundsor biomolecules that correlates with the stage of certain diseases,e.g., infectious and parasitic disease, injuries, cardiovasculardisease, cancer, mental disorders, neuropsychiatric disorders andorganic diseases, e.g., pulmonary diseases, renal diseases, (b) thedetection, purification and quantification of microorganism, e.g.,virus, fungus and bacteria from environment, e.g., water, soil, orbiological samples, e.g., tissues, bodily fluids, (c) the detection,quantification of chemical compounds or biological samples that posehazard to food safety or national security, e.g. toxic waste, anthrax,(d) quantification of vital parameters in medical or physiologicalmonitor, e.g., glucose, blood oxygen level, total blood count, (e) thedetection and quantification of specific DNA or RNA from biosamples,e.g., cells, viruses, bodily fluids, (f) the sequencing and comparing ofgenetic sequences in DNA in the chromosomes and mitochondria for genomeanalysis or (g) to detect reaction products, e.g., during synthesis orpurification of pharmaceuticals. Some of the specific applications ofthe devices, systems and methods in the present invention are describednow in further detail.

I) Diagnostic Method

In certain embodiments, the subject method finds use in detectingbiomarkers. In certain embodiments, the devices, systems and methods inthe present invention of using CROF are used to detect the presence orabsence of particular biomarkers, as well as an increase or decrease inthe concentration of particular biomarkers in blood, plasma, serum, orother bodily fluids or excretions, such as but not limited to urine,blood, serum, plasma, saliva, semen, prostatic fluid, nipple aspiratefluid, lachrymal fluid, perspiration, feces, cheek swabs, cerebrospinalfluid, cell lysate samples, amniotic fluid, gastrointestinal fluid,biopsy tissue, and the like. Thus, the sample, e.g. a diagnostic sample,can include various fluid or solid samples.

In some instances, the sample can be a bodily fluid sample from asubject who is to be diagnosed. In some instances, solid or semi-solidsamples can be provided. The sample can include tissues and/or cellscollected from the subject. The sample can be a biological sample.Examples of biological samples can include but are not limited to,blood, serum, plasma, a nasal swab, a nasopharyngeal wash, saliva,urine, gastric fluid, spinal fluid, tears, stool, mucus, sweat, earwax,oil, a glandular secretion, cerebral spinal fluid, tissue, semen,vaginal fluid, interstitial fluids derived from tumorous tissue, ocularfluids, spinal fluid, a throat swab, breath, hair, finger nails, skin,biopsy, placental fluid, amniotic fluid, cord blood, lymphatic fluids,cavity fluids, sputum, pus, microbiota, meconium, breast milk, exhaledcondensate and/or other excretions. The samples can includenasopharyngeal wash. Nasal swabs, throat swabs, stool samples, hair,finger nail, ear wax, breath, and other solid, semi-solid, or gaseoussamples can be processed in an extraction buffer, e.g., for a fixed orvariable amount of time, prior to their analysis. The extraction bufferor an aliquot thereof can then be processed similarly to other fluidsamples if desired. Examples of tissue samples of the subject caninclude but are not limited to, connective tissue, muscle tissue,nervous tissue, epithelial tissue, cartilage, cancerous sample, or bone.

In some instances, the subject from which a diagnostic sample isobtained can be a healthy individual, or can be an individual at leastsuspected of having a disease or a health condition. In some instances,the subject can be a patient.

In certain embodiments, the CROF device includes a capture agentconfigured to specifically bind a biomarker in a sample provided by thesubject. In certain embodiments, the biomarker can be a protein. Incertain embodiments, the biomarker protein is specifically bound by anantibody capture agent present in the CROF device. In certainembodiments, the biomarker is an antibody specifically bound by anantigen capture agent present in the CROF device. In certainembodiments, the biomarker is a nucleic acid specifically bound by anucleic acid capture agent that is complementary to one or both strandsof a double-stranded nucleic acid biomarker, or complementary to asingle-stranded biomarker. In certain embodiments, the biomarker is anucleic acid specifically bound by a nucleic acid binding protein. Incertain embodiments, the biomarker is specifically bound by an aptamer.

The presence or absence of a biomarker or significant changes in theconcentration of a biomarker can be used to diagnose disease risk,presence of disease in an individual, or to tailor treatments for thedisease in an individual. For example, the presence of a particularbiomarker or panel of biomarkers can influence the choices of drugtreatment or administration regimes given to an individual. Inevaluating potential drug therapies, a biomarker can be used as asurrogate for a natural endpoint such as survival or irreversiblemorbidity. If a treatment alters the biomarker, which has a directconnection to improved health, the biomarker can serve as a surrogateendpoint for evaluating the clinical benefit of a particular treatmentor administration regime. Thus, personalized diagnosis and treatmentbased on the particular biomarkers or panel of biomarkers detected in anindividual are facilitated by the subject method. Furthermore, the earlydetection of biomarkers associated with diseases is facilitated by thehigh sensitivity of the devices, systems and methods in the presentinvention, as described above. Due to the capability of detectingmultiple biomarkers with a mobile device, such as a smartphone, combinedwith sensitivity, scalability, and ease of use, the presently disclosedmethod finds use in portable and point-of-care or near-patient moleculardiagnostics.

In certain embodiments, the subject method finds use in detectingbiomarkers for a disease or disease state. In certain instances, thesubject method finds use in detecting biomarkers for thecharacterization of cell signaling pathways and intracellularcommunication for drug discovery and vaccine development. For example,the subject method can be used to detect and/or quantify the amount ofbiomarkers in diseased, healthy or benign samples. In certainembodiments, the subject method finds use in detecting biomarkers for aninfectious disease or disease state. In some cases, the biomarkers canbe molecular biomarkers, such as but not limited to proteins, nucleicacids, carbohydrates, small molecules, and the like.

The subject method find use in diagnostic assays, such as, but notlimited to, the following: detecting and/or quantifying biomarkers, asdescribed above; screening assays, where samples are tested at regularintervals for asymptomatic subjects; prognostic assays, where thepresence and or quantity of a biomarker is used to predict a likelydisease course; stratification assays, where a subject's response todifferent drug treatments can be predicted; efficacy assays, where theefficacy of a drug treatment is monitored; and the like.

In certain embodiments, a subject biosensor can be used diagnose apathogen infection by detecting a target nucleic acid from a pathogen ina sample. The target nucleic acid can be, for example, from a virus thatis selected from the group comprising human immunodeficiency virus 1 and2 (HIV-1 and HIV-2), human T-cell leukaemia virus and 2 (HTLV-1 andHTLV-2), respiratory syncytial virus (RSV), adenovirus, hepatitis Bvirus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), humanpapillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus(CMV), herpes-simplex virus 1 and 2 (HSV-1 and HSV-2), human herpesvirus8 (HHV-8, also known as Kaposi sarcoma herpesvirus) and flaviviruses,including yellow fever virus, dengue virus, Japanese encephalitis virus,West Nile virus and Ebola virus. The present invention is not, however,limited to the detection of nucleic acid, e.g., DNA or RNA, sequencesfrom the aforementioned viruses, but can be applied without any problemto other pathogens important in veterinary and/or human medicine.

Human papillomaviruses (HPV) are further subdivided on the basis oftheir DNA sequence homology into more than 70 different types. Thesetypes cause different diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29cause benign warts. HPV types 5, 8, 9, 12, 14, 15, 17 and 19-25 and46-50 cause lesions in patients with a weakened immune system. Types 6,11, 34, 39, 41-44 and 51-55 cause benign acuminate warts on the mucosaeof the genital region and of the respiratory tract. HPV types 16 and 18are of special medical interest, as they cause epithelial dysplasias ofthe genital mucosa and are associated with a high proportion of theinvasive carcinomas of the cervix, vagina, vulva and anal canal.Integration of the DNA of the human papillomavirus is considered to bedecisive in the carcinogenesis of cervical cancer. Humanpapillomaviruses can be detected for example from the DNA sequence oftheir capsid proteins L1 and L2. Accordingly, the method of the presentinvention is especially suitable for the detection of DNA sequences ofHPV types 16 and/or 18 in tissue samples, for assessing the risk ofdevelopment of carcinoma.

Other pathogens that can be detected in a diagnostic sample using thedevices, systems and methods in the present invention include, but arenot limited to: Varicella zoster, Staphylococcus epidermidis,Escherichia coli, methicillin-resistant Staphylococcus aureus (MSRA),Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis,Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococcus warneri,Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus simulans,Streptococcus pneumoniae and Candida albicans; gonorrhea (Neisseriagorrhoeae), syphilis (Treponena pallidum), clamydia (Clamydatracomitis), nongonococcal urethritis (Ureaplasm urealyticum), chancroid(Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis);Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus(MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphylococcusaureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,Escherichia coli, Enterococcus faecalis, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans,Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens,Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsella pneumoniae Legionella pneumophila, Mycoplasma pneumoniae, andMycobacterium tuberculosis, etc.

In some cases, the CROF device can be employed to detect a biomarkerthat is present at a low concentration. For example, the CROF device canbe used to detect cancer antigens in a readily accessible bodily fluids(e.g., blood, saliva, urine, tears, etc.), to detect biomarkers fortissue-specific diseases in a readily accessible bodily fluid (e.g., abiomarkers for a neurological disorder (e.g., Alzheimer's antigens)), todetect infections (particularly detection of low titer latent viruses,e.g., HIV), to detect fetal antigens in maternal blood, and fordetection of exogenous compounds (e.g., drugs or pollutants) in asubject's bloodstream, for example.

One potential source of the biomarker (e.g., “CSF”; cerebrospinal fluid)is also indicated in the table. In many cases, the subject biosensor candetect those biomarkers in a different bodily fluid to that indicated.For example, biomarkers that are found in CSF can be identified inurine, blood or saliva. It will also be clear to one with ordinary skillin the art that the subject CROF devices can be configured to captureand detect many more biomarkers known in the art that are diagnostic ofa disease or health condition.

A biomarker can be a protein or a nucleic acid (e.g., mRNA) biomarker,unless specified otherwise. The diagnosis can be associated with anincrease or a decrease in the level of a biomarker in the sample, unlessspecified otherwise. Lists of biomarkers, the diseases that they can beused to diagnose, and the sample in which the biomarkers can be detectedare described in Tables 1 and 2 of U.S. provisional application Ser. No.62/234,538, filed on Sep. 29, 2015, which application is incorporated byreference herein.

In some instances, the devices, systems and methods in the presentinvention is used to inform the subject from whom the sample is derivedabout a health condition thereof. Health conditions that can bediagnosed or measured by the devices, systems and methods in the presentinvention, device and system include, but are not limited to: chemicalbalance; nutritional health; exercise; fatigue; sleep; stress;prediabetes; allergies; aging; exposure to environmental toxins,pesticides, herbicides, synthetic hormone analogs; pregnancy; menopause;and andropause. Table 3 of U.S. provisional application Ser. No.62/234,538, filed on Sep. 29, 2015, which application is incorporated byreference herein, provides a list of biomarker that can be detectedusing the present CROF device (when used in conjunction with anappropriate monoclonal antibody, nucleic acid, or other capture agent),and their associated health conditions.

J) Kits

Aspects of the present disclosure include a kit that find use inperforming the devices, systems and methods in the present invention, asdescribed above. In certain embodiments, the kit includes instructionsfor practicing the subject methods using a hand held device, e.g., amobile phone. These instructions can be present in the subject kits in avariety of forms, one or more of which can be present in the kit. Oneform in which these instructions can be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, etc. Another means would be a computerreadable medium, e.g., diskette, CD, DVD, Blu-Ray, computer-readablememory, etc., on which the information has been recorded or stored. Yetanother means that can be present is a website address which can be usedvia the Internet to access the information at a removed site. The kitcan further include a software for implementing a method for measuringan analyte on a device, as described herein, provided on a computerreadable medium. Any convenient means can be present in the kits.

In certain embodiments, the kit includes a detection agent that includesa detectable label, e.g. a fluorescently labeled antibody oroligonucleotide that binds specifically to an analyte of interest, foruse in labeling the analyte of interest. The detection agent can beprovided in a separate container as the CROF device, or can be providedin the CROF device. In certain embodiments, the kit includes a controlsample that includes a known detectable amount of an analyte that is tobe detected in the sample. The control sample can be provided in acontainer, and can be in solution at a known concentration, or can beprovided in dry form, e.g., lyophilized or freeze dried. The kit canalso include buffers for use in dissolving the control sample, if it isprovided in dry form.

EXAMPLES A) Example 1

An OAC is a QMAX device having two plates.

The first plate is a rectangle shaped PMMA plate with a flat surface anda thickness in the range of 0.8 mm to 1.1 mm, 0.5 mm to 1.5 mm, or 0.3mm to 2 mm; a length in the range of 28 mm to 32 mm, 25 mm to 35 mm, or20 mm to 50 mm; and a width in the range of 20 mm to 28 mm, 15 mm to 34mm, or 10 mm to 40 mm.

The second plate is a rectangle shaped PMMA film with a flat surface andan array of micro pillar array imprinted on the flat surface. The PMMAfilm has a thickness in the range of 0.8 mm to 1.1 mm, 0.5 mm to 1.5 mm,or 0.3 mm to 2 mm; a length in the range of 28 mm to 32 mm, 25 mm to 35mm, or 20 mm to 50 mm; and a width in the range of 20 mm to 28 mm, 15 mmto 34 mm, or 10 mm to 40 mm. In some embodiments, when putting the firstplate and the second plate together to hold sample, at least three sidesof the second plate is inside the area of the first plate. The pillararray has a shape of either rectangle or square, a flat top, and apillar lateral size in the range of 30 um to 40 um, 25 um to 45 um, 20,um to 50 um, 10 um to 60 um, or 5 um to 70 um, a pillar height in therange of 10 um to 30 um, 5 um to 40 um, 1 um to 50 um, or0.1 um to 100um, and a distance between two neighboring pillar center in a range of80 to 110 um, 60 to 130 um, or 30 to 180 um, or 30 to 200 um.

B) Hemoglobin Measurements Using OAC—Using One Wavelength

In an experiment of the present invention, an OAC is a QMAX device hastwo plates. The first plate is 1 mm thick flat PMMA substrate with asize of 30 mm×24 mm. The second plate is 175 um thick PMMA film with amicro pillar array on it with a size of 24 mm×22 mm. The pillar arrayhas pillar size of 30 um×40 um, pillar to pillar edge distance of 80 um,and pillar height of 10 um or 30 um.

The sample is a fresh whole blood (2.5 uL for 10 um pillar height, 5 uLfor 30 um pillar height), which was dropped in a location of the firstplate, and pressed by the second plate.

In the optical measurements, as shown in FIG. 5, LED light are filteredby a band pass filter (532 nm to 576 nm) and illuminates onto two 45degree mirror sets. The light then goes through a semi-opaque diffuser,to eliminate coherence of the point source's wavefront and ensure theintensity change is only due to the absorption. Finally, the lighttransmits the QMAX device and is collected by a lens and camera.

The LED light and camera used here can be both from a phone.

The picture taken by camera shows that there are 2 regions. One regionis the pillar region, the other is the blood region.

The absorption of light in pillar region is neglectable. Also, theextinction coefficient of oxygenated hemoglobin [HbO2] and deoxygenatedhemoglobin [Hb] in wavelength range of 532 nm to 576 nm is similarε_(Hb)≈ε_(HbO) ₂ =44000-48000 cm⁻¹/M.

Thus, OD^(green)=ln(I/Io)=ε_(HbO) ₂ ^(greem){[Hb]+[HbO₂]}L

As shown in FIG. 6, I is the average intensity in the blood region, Iois the average intensity in the center of the pillar region. Whencalculating the average intensity, we subtract 5 um area near the pillarboundary to reduce the analyze error.

${{Total}\mspace{14mu} {hemoglobin}\mspace{14mu} {concentration}} = {{\left\lbrack {HbO}_{2} \right\rbrack + \lbrack{Hb}\rbrack} = \frac{\ln \left( \frac{I}{Io} \right)}{ɛ \times {gap}}}$

We measured the hemoglobin in blood ranging from 6 g/dL to 11 g/dL withboth QMAX device setup and commercial Abbott Emerald hemocytometermachine and compared the results as shown in Figure. 7. For eachconcentration, we measured 3 cards to calculate the standard deviation.

From the results, the repeatability (CV) of hemoglobin measurements byQMAX card for same blood is around 5% and the R2 value compared withgold standard is 96%.

C) Example-3

FIG. 8. illustrates an example of a hemoglobin measurement, where theimage of an optical transmission image through a thin layer of wholeblood (without lysing) in an OAC (e.g. the sample holder described inFIG. 1), and the light source is a diffusive light source (e.g. a lightdiffusion is placed in front of a point light source), and the imagetaken by an iPhone. In FIG. 8, the light-guiding spacers areperiodically placed on a QMAX card, with a vertical periodic distance of120 um and a horizontal periodic distance of 110 um.

FIG. 9 illustrates an example of the sample regions and referenceregions selected for determining hemoglobin using the image from FIG. 8.The boundary of the sampling regions and the reference are marked.

In FIG. 9, the reference regions (with outer boundaries colored in blue)are inside light-guiding spacers; D, the distance between the edges ofthe reference region and the light-guiding spacer, is 10 um; d, thedistance between the edges of the sampling region and the light-guidingspacer, is 30 um; and T, the distance between the edges of the samplingregion and the reference region, is 40 um.

D) Image Processing

According to the present invention, the image processing algorithm onhemoglobin absorption measurement consists of the following steps:

1. Light-guiding spacer detection

2. Reference region and sampling region determination

3. Individual region calculation

4. Polling

The light-guiding spacer detection is to detect and locate light-guidingspacers, which are periodically placed on a QMAX card. Various objectdetection algorithm can be employed, including, but not limited to,template matching, blob detection, contour detection, etc. The detectioncan be performance in a single color channel in a color space (RGB, HSV,HSI, Lab, YCrCb, etc.), such as the green channel in a RGB color space,or the hue channel in the HSV space, or a combination of two or morecolor channels, such as using red-green-blue channels in a RGB colorspace, etc.

After the periodic light-guiding spaces are detected and located, thereference regions (which are inside the light-guiding spacers) andsampling regions are selected. The sizes of reference regions andsampling regions, and the distance among the edges of the light-guidingspaces, reference regions, and sampling regions are disclosed in theinvention.

For reference regions and sampling regions, they can be associated bythe relative location and distance. When one (or more) reference regionis associated with one (or more) sampling region, a hemoglobinabsorption measurement can be calculated by the method disclosed in theinvention. One embodiment is to associate one reference region with thesampling region with the shortest distance, and calculate the hemoglobinabsorption measurement for each association.

A pooling algorithm is to pool hemoglobin absorption measurements fromeach associate and produce a single hemoglobin absorption measurement.Various pooling algorithm can be utilized, such as median, mean, max,min, k-means, etc.

In some embodiments, the imaging processing uses artificial intelligenceand/or machine learning. In some embodiments, the imaging processinguses deep learning.

E) Using Two Wavelengths

Similar to above experimental setup, except using 2 different band passfilters and taking 2 pictures.

After taking the picture, by calculating the

${OD} = {\ln \left( \frac{I}{Io} \right)}$

of blood with two different wavelength λ₁ and λ₂, e.g, 660 nm and 940nm:

OD^(λ) ¹ ={ε_(Hb) ^(λ) ¹ [Hb]+ε_(HbO) ₂ ^(λ) ¹ [HbO₂]}L

OD^(λ) ² ={ε_(Hb) ^(λ) ² [Hb]+ε_(HbO) ₂ ^(λ) ² [HbO₂]}L

We get:

$\left\lbrack {HbO}_{2} \right\rbrack = {{\frac{{ɛ_{Hb}^{\lambda_{2}}{OD}^{\lambda_{1}}} - {ɛ_{Hb}^{\lambda_{1}}{OD}^{\lambda_{2}}}}{L\left( {{ɛ_{Hb}^{\lambda_{2}}ɛ_{{HbO}_{2}}^{\lambda_{1}}} - {ɛ_{Hb}^{\lambda_{1}}ɛ_{{HbO}_{2}}^{\lambda_{2}}}} \right)}\lbrack{Hb}\rbrack} = \frac{{ɛ_{{HbO}_{2}}^{\lambda_{2}}{OD}^{\lambda_{1}}} - {ɛ_{{HbO}_{2}}^{\lambda_{1}}{OD}^{\lambda_{2}}}}{L\left( {{ɛ_{Hb}^{\lambda_{1}}ɛ_{{HbO}_{2}}^{\lambda_{2}}} - {ɛ_{Hb}^{\lambda_{2}}ɛ_{{HbO}_{2}}^{\lambda_{1}}}} \right)}}$

ε is the extinction coefficient of hemoglobin, [Hb] and [HbO₂] is theconcentration of hemoglobin, and L is the length of light path throughthe sample or the gap size of QMAX device.

Thus, total hemoglobin concentration=[HbO₂]+[Hb].

This method could further provide the detailed information of the ratiobetween [HbO2] and [Hb].

F) Light Guiding Spacer, Sampling Region, and Reference Region

In some embodiments, the sampling region boundary has a size of 120 umby 110 um; the edge of sampling area has a size of 60 um by 45 um; thelight guiding spacer or pillar has a size of 40 um by 30 um; thereference region has a size of 20 um by 15 um. In some embodiments, thearea of reference region is 1/2 of the size of the light guiding spacerarea, the distance between edge of the sampling area and that of thelight guiding spacer is 1/2 of the light guiding spacer area, and thearea of the sampling area is equal to the periodic inter spacerdistance.

-   1. The method or device of any prior claim, wherein the spacers have    pillar shape and nearly uniform cross-section.-   2. The method or device of any prior claim, wherein the inter spacer    distance (SD) is equal or less than about 150 um (micrometer).-   3. The method or device of any prior claim, wherein the inter spacer    distance (SD) is equal or less than about 100 um (micrometer).-   4. The method or device of any prior claim, wherein the fourth power    of the inter-spacer-distance (ISD) divided by the thickness (h) and    the Young's modulus (E) of the flexible plate (ISD⁴/(hE)) is 5×10⁶    um³/GPa or less.-   5. The method or device of any prior claim, wherein the fourth power    of the inter-spacer-distance (ISD) divided by the thickness (h) and    the Young's modulus (E) of the flexible plate (ISD⁴/(hE)) is 5×10⁵    um³/GPa or less.-   6. The method or device of any prior claim, wherein the spacers have    pillar shape, a substantially flat top surface, a predetermined    substantially uniform height, and a predetermined constant    inter-spacer distance that is at least about 2 times larger than the    size of the analyte, wherein the Young's modulus of the spacers    times the filling factor of the spacers is equal or larger than 2    MPa, wherein the filling factor is the ratio of the spacer contact    area to the total plate area, and wherein, for each spacer, the    ratio of the lateral dimension of the spacer to its height is at    least 1 (one).-   7. The method or device of any prior claim, wherein the spacers have    pillar shape, a substantially flat top surface, a predetermined    substantially uniform height, and a predetermined constant    inter-spacer distance that is at least about 2 times larger than the    size of the analyte, wherein the Young's modulus of the spacers    times the filling factor of the spacers is equal or larger than 2    MPa, wherein the filling factor is the ratio of the spacer contact    area to the total plate area, and wherein, for each spacer, the    ratio of the lateral dimension of the spacer to its height is at    least 1 (one), wherein the fourth power of the inter-spacer-distance    (ISD) divided by the thickness (h) and the Young's modulus (E) of    the flexible plate (ISD⁴/(hE)) is 5×10⁶ um³/GPa or less.-   8. The device of any prior device claim, wherein the ratio of the    inter-spacing distance of the spacers to the average width of the    spacer is 2 or larger, and the filling factor of the spacers    multiplied by the Young's modulus of the spacers is 2 MPa or larger.-   9. The method or device of any prior claim, wherein the analytes is    the analyte in 5 detection of proteins, peptides, nucleic acids,    synthetic compounds, and inorganic compounds.-   10. The method or device of any prior claim, wherein the sample is a    biological sample selected from amniotic fluid, aqueous humour,    vitreous humour, blood (e.g., whole blood, fractionated blood,    plasma or serum), breast milk, cerebrospinal fluid (CSF), cerumen    (earwax), chyle, chime, endolymph, perilymph, feces, breath, gastric    acid, gastric juice, lymph, mucus (including nasal drainage and    phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus,    rheum, saliva, exhaled breath condensates, sebum, semen, sputum,    sweat, synovial fluid, tears, vomit, and urine.-   11. The method or device of any prior claim, wherein the spacers    have a shape of pillars and a ratio of the width to the height of    the pillar is equal or larger than one.-   12. The method of any prior claim, wherein the sample that is    deposited on one or both of the plates has an unknown volume.-   13. The method or device of any prior claim, wherein the spacers    have a shape of pillar, and the pillar has substantially uniform    cross-section.-   14. The method or device of any prior claim, wherein the samples is    for the detection, purification and quantification of chemical    compounds or biomolecules that correlates with the stage of certain    diseases.-   15. The method or device of any prior claim, wherein the samples is    related to infectious and parasitic disease, injuries,    cardiovascular disease, cancer, mental disorders, neuropsychiatric    disorders, pulmonary diseases, renal diseases, and other and organic    diseases.-   16. The method or device of any prior claim, wherein the samples is    related to the detection, purification and quantification of    microorganism.-   17. The method or device of any prior claim, wherein the samples is    related to virus, fungus and bacteria from environment, e.g., water,    soil, or biological samples.-   18. The method or device of any prior claim, wherein the samples is    related to the detection, quantification of chemical compounds or    biological samples that pose hazard to food safety or national    security, e.g. toxic waste, anthrax.-   19. The method or device of any prior claim, wherein the samples is    related to quantification of vital parameters in medical or    physiological monitor.-   20. The method or device of any prior claim, wherein the samples is    related to glucose, blood, oxygen level, total blood count.-   21. The method or device of any prior claim, wherein the samples is    related to the detection and quantification of specific DNA or RNA    from biosamples.-   22. The method or device of any prior claim, wherein the samples is    related to the sequencing and comparing of genetic sequences in DNA    in the chromosomes and mitochondria for genome analysis.-   23. The method or device of any prior claim, wherein the samples is    related to detect reaction products, e.g., during synthesis or    purification of pharmaceuticals.-   24. The method or device of any prior claim, wherein the samples is    cells, tissues, bodily fluids, and stool.-   25. The method or device of any prior claim, wherein the sample is    the sample in the detection of proteins, peptides, nucleic acids,    synthetic compounds, inorganic compounds.-   26. The method or device of any prior claim, wherein the sample is    the sample in the fields of human, veterinary, agriculture, foods,    environments, and drug testing.    The method or device of any prior claim, wherein the sample is a    biological sample is selected from blood, serum, plasma, a nasal    swab, a nasopharyngeal wash, saliva, urine, gastric fluid, spinal    fluid, tears, stool, mucus, sweat, earwax, oil, a glandular    secretion, cerebral spinal fluid, tissue, semen, vaginal fluid,    interstitial fluids derived from tumorous tissue, ocular fluids,    spinal fluid, a throat swab, breath, hair, finger nails, skin,    biopsy, placental fluid, amniotic fluid, cord blood, lymphatic    fluids, cavity fluids, sputum, pus, microbiota, meconium, breast    milk, exhaled condensate nasopharyngeal wash, nasal swab, throat    swab, stool samples, hair, finger nail, ear wax, breath, connective    tissue, muscle tissue, nervous tissue, epit

Other Descriptions and Additional Examples of Present Inventions

The present invention comprises further a combination of the disclosuresabove together a variety of embodiments that are given in the below,which can be combined in multiple ways as long as the various componentsdo not contradict one another. The embodiments should be regarded as asingle invention file: each filing has other filing as the referencesand is referenced in its entirety and for all purpose, rather than as adiscrete independent. These embodiments include not only the disclosuresin the current file, but the documents that are herein referenced,incorporated, or to which priority is claimed.

A device for analyzing an analyte in a sample using opticaltransmission, comprising:

a first plate, a second plate, and light-guiding spacers (LGS's),wherein:

the first plate and second plate are movable relative to each other intodifferent configurations, including an open configuration and a closedconfiguration;

each of the plates comprises an inner surface that has a sample contactarea for contacting a sample that contains or is suspected to containanalyte; and

each of the light-guiding spacers has a pillar shape, has bottom endfixed on one of the plate and the top end having flat surface, whereinthe light-guiding spacers have a uniform height of 300 um (microns) orless, wherein, in a closed configuration, the top end flat surface is indirect contact with the plate, and the cross-section of eachlight-guiding spacer is larger than the wavelength of the light thatanalyzes the sample,

wherein at least one of the light-guiding spacer is inside the sample;

-   -   wherein the open configuration is a configuration, the two        plates are separated apart, the spacing between the plates is        not regulated by the light-guiding spacers, and the sample is        deposited on one or both of the plates;    -   wherein the closed configuration is a configuration, which is        configured after the sample deposition in the open        configuration; at least part of the sample is compressed by the        two plates into a layer of uniform thickness, wherein the        uniform thickness of the layer is confined by the t plates and        is regulated by the plates and the light-guiding spacers; and

A device for analyzing an analyte in a sample using opticaltransmission, comprising:

-   -   a first plate, a second plate, a light guiding spacer (LGS) or        more, a sampling region, and a reference region, wherein:

the first plate and second plate are configured to sandwich a sample,this is for an optical transmission analysis by light, into a thin layerbetween the plates, and each plate has a sample contact area on itsinner surface that contacts the sample;

each of the light-guiding spacer (LGS) or more LGS's has a pillar shape,is sandwiched between the two plates with each end of the pillar indirect contact to one of the plates forming a LGS-plate contact area,and is configured to allow the light transmits from the first plate,through the LGS, to the second plate without going through a sample,

the sampling region is the region that the light can go through, insequence, the first plate, the sample, and the second plate, wherein thesampling region does not have the LGS; and

the reference region is the region that the light transmits through, insequence, the first plate, the light-guiding spacer, and the secondplate, without going through the sample;

-   -   wherein the LGS-contact areas and a lateral cross-section of the        LGS are larger than the wavelength of the light,    -   wherein the light-guiding spacer is surrounded by or near the        sample; and    -   wherein the sample in the sampling region has a thickness of 300        um or less.    -   LGSs are an periodic array    -   LGSs are an periodic array and the period is 500 um or less.    -   LGSs are an periodic array and the period is 250 um or less.    -   LGSs are an periodic array and the period is 150 um or less.        -   the height is 60 um or less        -   the height is 40 um or less.    -   in the closed configuration: (a) at least one spacer in the        sample contact area has its top surface in direct contact with        one of the plates, and the at least one spacer and the regions        of the plates above and below the at least one spacer define a        reference region wherein the reference region is transparent to        light within a wavelength range, and (b) at least one region in        the sample contact area on one plate and its corresponding        region on the other plate are not occupied by the light-guiding        spacers, defining a sampling region that is transparent to light        within the same wavelength range.

A device for analyzing an analyte in a sample using opticaltransmission, comprising:

a first plate, a second plate, and a light-guiding spacer, wherein:

the first plate and second plate are configured to hold a sample thatcontains or is suspected to contain an analyte, wherein at least partthe sample is between the two plates and is in contact with both plates;and

the light-guiding spacer has a pillar shape and a predeterminedsubstantially height, wherein the light-guiding spacer is surrounded bythe sample during an optical transmission measurement;

-   -   wherein top and bottom surfaces of the light-guiding spacers are        substantially flat and the top and bottom surfaces of the light        guiding spacer is in direct contact with the plates,    -   wherein the area of the top and bottom surfaces and average        lateral cross-section of each spacer is respectively larger than        the wavelength of the light that analyze the sample, and

wherein: (a) the at least one spacer and the regions of the platesdirectly above and below the at least one spacer define a referenceregion that is transparent to light within a wavelength range andpassing through the plates and the spacer, and (b) at least one regionin the sample contact area on one plate and its corresponding region onthe other plate are not occupied by the light-guiding spacers, defininga sampling region that is transparent to light within the samewavelength range.

A device for analyzing an analyte in a sample, comprising:

a first plate, a second plate, and light-guiding spacers, wherein:

the first plate and second plate are movable relative to each other intodifferent configurations, including an open configuration and a closedconfiguration;

each of the plates comprises an inner surface that has a sample contactarea for contacting a sample that contains or is suspected to contain ananalyte; and

the light-guiding spacers have a pillar shape and a predeterminedsubstantially uniform height, wherein top and bottom surfaces of thelight-guiding spacers are substantially flat and the bottom surface isfixed on the inner surface of one or the plates, wherein the area of thetop and bottom surfaces and average lateral cross-section of each spaceris respectively larger than 1 the wavelength of the light that analyzethe sample, wherein at least one of the light-guiding spacers is insidethe sample contact area;

wherein the open configuration is a configuration, in which: the twoplates are separated apart, the spacing between the plates is notregulated by the light-guiding spacers, and the sample is deposited onone or both of the plates;

wherein the closed configuration is aa configuration, which isconfigured after the sample deposition in the open configuration; and inthe closed configuration: at least part of the sample is compressed bythe two plates into a layer of highly uniform thickness, wherein theuniform thickness of the layer is confined by the sample contact areasof the plates and is regulated by the plates and the light-guidingspacers; and

wherein in the closed configuration, (a) at least one spacer in thesample contact area has its top surface in direct contact with one ofthe plates, and the at least one spacer and the regions of the platesabove and below the at least one spacer define a reference regionwherein the reference region is transparent to light within a wavelengthrange, and (b) at least one region in the sample contact area on oneplate and its corresponding region on the other plate are not occupiedby the light-guiding spacers, defining a sampling region that istransparent to the light within the wavelength range.

A device for analyzing tin a sample, comprising:

a first plate, a second plate, and a light guiding spacer, wherein:

the first plate and second plate are configured to sandwich a sampleinto a thin layer;

a first plate, a second plate, and a light guiding spacer, wherein:

the first plate and second plate are configured to sandwich a sampleinto a thin layer;

the light-guiding spacer has a pillar shape that is sandwiched betweenthe two plates,

wherein each end of the pillar directly contact one of the plates sothat there is no sample between the end of the pillar and the respectiveplate, wherein the light-guiding spacer is either surrounded by or nearthe sample, and wherein the direct-contact area and an average lateralcross-section of the pillar is respectively at least 1 um{circumflexover ( )}2 (square micron) or larger;

wherein the spacing between the inner surfaces of the plates is 200 umor less.

An apparatus for sample analysis using optical transmission, comprising:

a device of any of prior device embodiments, a light source, a camera;and an adaptor, wherein

the light source is configured to emit light in the wavelength rangethat is configured to go through the reference region;

the camera is configured to image the reference region and the samplingregion. the adaptor is configured to position the device, the lightsource, and the camera relative to each other, so that the light fromthe light source goes through the reference region and the samplingregion and is imaged by the camera.

The apparatus of any prior apparatus embodiments, further comprising:

a processor, which is configured to process the images captured by thecamera, and

determine a property of the analyte in the sample based on comparinglight transmissions from the reference region and the sampling region.

The apparatus of any prior claim, wherein the camera and the processorare parts of a single mobile device.

The apparatus of any prior claim, wherein the light source and theprocessor are parts of a single mobile device.

The apparatus of any prior claim, wherein the light source, the camera,and the processor are parts of a single mobile device.

The apparatus of any prior apparatus embodiments, wherein the mobiledevice is a smart phone.

A method for sample analysis using an transmitted light, comprising thesteps of: having a device of any prior device embodiments;

depositing the sample at the open configuration of the device, whereinthe sample is suspected of containing an analyte;

bringing the device into the closed configuration;

having a light source that has a wavelength that is configured to gothrough the reference region of the device;

having an imager that is configured to image the reference region andthe sampling region of the device;

having an adaptor that is configured to position the device, the lightsource, and the camera relative to each other, so that the light fromthe light source goes through the reference region and the samplingregion and is imaged by the camera;

determining a property of the analyte by comparing the lighttransmission from the sampling region and the reference region.

The device, method, or system of any prior claim, wherein the analyte ishemoglobin.

The device, method, or system of any prior claim, wherein the analyte istype of cells.

Add all analytes.

Add different applications.

Add key distances between the light transmission.

The device, method, or system of any prior claim, wherein the thicknessof the sample layer is regulated by plates and the light-guiding spacersand is substantially the same as the uniform height of the light-guidingspacers;

The device, method, or system of any prior claim, wherein the analyte isred blood cells.

The device, method, or system of any prior claim, wherein the analyte iswhite blood cells.

The device, method, or system of any prior claim, wherein the referenceregion and the sampling region have a same size.

The device, method, or system of any prior claim, wherein the referenceregion is within a corresponding area of the cross section of thelight-guide spacer.

The device, method, or system of any prior claim, wherein the referenceregion is less than 0.1 um{circumflex over ( )}2, less than 0.2um{circumflex over ( )}2, less than 0.5 um{circumflex over ( )}2, lessthan 1 um{circumflex over ( )}2, less than 2 um{circumflex over ( )}2,less than 5 um{circumflex over ( )}2, less than 10 um{circumflex over( )}2, less than 20 um{circumflex over ( )}2, less than 50 um{circumflexover ( )}2, less than 100 um{circumflex over ( )}2, less than 200um{circumflex over ( )}2, less than 500 um{circumflex over ( )}2, lessthan 1000 um{circumflex over ( )}2, less than 2000 um{circumflex over( )}2, less than 5000 um{circumflex over ( )}2, less than 10000um{circumflex over ( )}2, less than 20000 um{circumflex over ( )}2, lessthan 50000 um{circumflex over ( )}2, less than 100000 um{circumflex over( )}2, less than 200000 um{circumflex over ( )}2, less than 500000um{circumflex over ( )}2, less than 1 mm{circumflex over ( )}2, lessthan 2 mm{circumflex over ( )}2, less than 5 mm{circumflex over ( )}2,less than 10 mm{circumflex over ( )}2, less than 20 mm{circumflex over( )}2, or less than 50 mm{circumflex over ( )}2, or in a range betweenany of the two values.

The device, method, or system of any prior claim, wherein the devicefurther comprises a plurality of light guiding spacers that havesubstantially uniform height, and wherein at least one of thelight-guiding spacers is inside the sample contact area.

The device, method, or system of any prior claim, wherein the devicefurther comprises a plurality of light guiding spacers that havesubstantially uniform height, wherein the distance between twoneighboring light guiding spacers are known, and wherein at least one ofthe light-guiding spacers is inside the sample contact area.

The device, method, or system of any prior claim, wherein the devicefurther comprises a plurality of light guiding spacers that havesubstantially uniform height, wherein the distances between twoneighboring light guiding spacers are known and are substantiallyconstant (i.e. the light guiding spacers are substantially a periodicarray), and wherein at least one of the light-guiding spacers is insidethe sample contact area.

The device, method, or system of any prior claim, wherein the bottomsurface of the light guiding spacer is fixed on the inner surface of oneof the plates by molding the light guiding spacer on the inner surfaceof the plate.

The device, method, or system of any prior claim, wherein the bottomsurface of the light guiding spacer is fixed on the inner surface of oneof the plates and is made of the same material as the inner surface.

The device, method, or system of any prior claim, wherein the bottomsurface of the light guiding spacer is fixed on the inner surface of oneof the plates, and is made of the same material as the inner surface,and the bottom surface of the light guiding spacer has no interface withon the inner surface of the plate.

The device, method, or system of any prior claim, wherein the wavelengthof the light is longer than 300 nm, and wherein the wavelength of thelight is also less than 20 μm, less than 15 μm, less than 10 μm, lessthan 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, less than 1μm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450nm, less than 400 nm, or in a range between any of the two values.

The device, method, or system of any prior claim, wherein the wavelengthof the light is longer than 500 nm, and wherein the wavelength of thelight is also less than 600 nm, less than 590 nm, less than 580 nm, lessthan 570 nm, less than 560 nm, less than 550 nm, less than 540 nm, lessthan 530 nm, less than 520 nm, less than 510 nm, or in a range betweenany of the two values.

The device, method, or system of any prior claim, wherein the averagelateral cross-section of each light-guiding spacer is less than 1um{circumflex over ( )}2 (micron-square), 10 um{circumflex over ( )}2,20 um{circumflex over ( )}2, 30 um{circumflex over ( )}2, 50um{circumflex over ( )}2, 100 um{circumflex over ( )}2, 150um{circumflex over ( )}2, 200 um{circumflex over ( )}2, 300um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, 100,000 um{circumflex over ( )}2, 200,000um{circumflex over ( )}2, 500,000 um{circumflex over ( )}2, 1mm{circumflex over ( )}2, 2 mm{circumflex over ( )}2, 5 mm{circumflexover ( )}2, 10 mm{circumflex over ( )}2, 50 mm{circumflex over ( )}2, orin a range between any of the two values.

The device, method, or system of any prior claim, wherein the averagelateral cross-section of each light-guiding spacer is less than 1um{circumflex over ( )}2 (micron-square), 10 um{circumflex over ( )}2,20 um{circumflex over ( )}2, 30 um{circumflex over ( )}2, 50um{circumflex over ( )}2, 100 um{circumflex over ( )}2, 150um{circumflex over ( )}2, 200 um{circumflex over ( )}2, 300um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, 100,000 um{circumflex over ( )}2, 200,000um{circumflex over ( )}2, or in a range between any of the two values.

The device, method, or system of any prior claim, wherein the averagelateral cross-section of each light-guiding spacer is less than 1um{circumflex over ( )}2 (micron-square), 10 um{circumflex over ( )}2,20 um{circumflex over ( )}2, 30 um{circumflex over ( )}2, 50um{circumflex over ( )}2, 100 um{circumflex over ( )}2, 150um{circumflex over ( )}2, 200 um{circumflex over ( )}2, 300um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, or in a range between any of the two values.

The device, method, or system of any prior claim, wherein the samplecontact area is larger than 100 um{circumflex over ( )}2(micron-square),larger than 200 um{circumflex over ( )}2, larger than 400 um{circumflexover ( )}2, larger than 600 um{circumflex over ( )}2, larger than 800um{circumflex over ( )}2, larger than 1,000 um{circumflex over ( )}2,larger than 2,000 um{circumflex over ( )}2, larger than 4,000um{circumflex over ( )}2, larger than 6,000 um{circumflex over ( )}2,larger than 8,000 um{circumflex over ( )}2, larger than 10,000um{circumflex over ( )}2, larger than 20,000 um{circumflex over ( )}2,larger than 40,000 um{circumflex over ( )}2, larger than 60,000um{circumflex over ( )}2, larger than 80,000 um{circumflex over ( )}2,larger than 100,000 um{circumflex over ( )}2, larger than 200,000um{circumflex over ( )}2, larger than 250,000 um{circumflex over ( )}2,larger than 500,000 um{circumflex over ( )}2(micron-square), or in arange between any of the two values.

The device, method, or system of any prior claim, wherein apredetermined constant inter-spacer distance that is at least about 2times larger than the size of the analyte.

The device, method, or system of any prior claim, wherein apredetermined constant inter-spacer distance that is larger than thesize of the analyte by a factor that is at least 2 times, at least 6times, at least 8 times, at least 10 times, at least 20 times, at least40 times, at least 60 times, at least 80 times, or at least 100 times.

The device, apparatus, or method of any prior claim, wherein the analyteis a biomarker, an environmental marker, or a foodstuff marker.

The device, apparatus, or method of any prior claim, wherein the analyteis a biomarker indicative of the presence or severity of a disease orcondition.

The device, apparatus, or method of any prior claim, wherein the analyteis a cell, a protein, or a nucleic acid.

The device, apparatus, or method of any prior claim, wherein the analyteis hemoglobin.

The device, apparatus, or method of any prior claim, wherein the analytecomprises proteins, peptides, nucleic acids, synthetic compounds,inorganic compounds, organic compounds, bacteria, virus, cells, tissues,nanoparticles, and other molecules, compounds, mixtures and substancesthereof.

The device, apparatus, or method of any prior claim, wherein the sampleis original, diluted, or processed forms of: bodily fluids, stool,amniotic fluid, aqueous humour, vitreous humour, blood, whole blood,fractionated blood, plasma, serum, breast milk, cerebrospinal fluid,cerumen, chyle, chime, endolymph, perilymph, feces, gastric acid,gastric juice, lymph, mucus, nasal drainage, phlegm, pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen,sputum, sweat, synovial fluid, tears, vomit, urine, or exhaled breathcondensate.

The device, apparatus, or method of any prior claim, wherein the sampleis original, diluted, or processed forms of blood.

The device, apparatus, or method of any prior claim, wherein the samplecomprises whole blood.

The device, apparatus, or method of any prior claim, wherein the samplecomprises an aggregation agent that induces aggregation of theinterference elements.

The device, apparatus, or method of any prior claim, wherein the sampleholder comprises wells that configured to hold the sample.

The device, apparatus, or method of any prior claim, wherein the sampleholder comprises a first plate, and a second plate, and spacers.

The device, apparatus, or method of any prior claim, wherein the sampleholder comprises a first plate, a second plate, and spacers, wherein thespacers are configured to regulate a gap between the plates when theplates are pressed against each, compressing the sample into a thinlayer.

The device, apparatus, or method of any prior claim, wherein the sampleholder comprises a first plate, a second plate, and spacers, andwherein:

-   -   the plates are moveable relative to each other into different        configurations, including an open configuration and a closed        configuration;    -   in the open configuration: the two plates are separated apart,        the spacing between the plates is not regulated by the spacers,        and the sample is deposited on one or both of the plates; and    -   in the closed configuration, which is configured after the        sample deposition in the open configuration: at least part of        the sample is compressed by the two plates into a layer of        highly uniform thickness and is substantially stagnant relative        to the plates, wherein the uniform thickness of the layer is        regulated by the plates and the spacers.

The device, apparatus, or method of any prior claim, wherein the sampleholder comprises a Q-card, which comprises a first plate, a secondplate, and spacers, wherein the spacers are configured to regulate a gapbetween the plates when the plates are pressed against each, compressingthe sample into a thin layer.

The device, apparatus, or method of any prior claim, wherein the sampleholder comprises a first plate, a second plate, and spacers, wherein thespacers have a uniform height and a constant inter-spacer distance; and

-   -   ii. the sample is compressed by the sample holder into a thin        layer with a uniform thickness that is regulated by the height        of the spacers.

The device, apparatus, or method of any prior claim, wherein the sampleis compressed into a layer of uniform thickness that substantiallyequals uniform height of spacers that are fixed to one or both of theplates.

The apparatus, kit or method of any prior claim, wherein the sample iscompressed into a layer of uniform thickness that has a variation ofless than 15%, 10%, 5%, 2%, 1%, or in a range between any of the twovalues.

The device, apparatus, or method of any prior claim, wherein the sample,when compressed, has a thickness of 500 nm or less, 1000 nm or less, 2μm (micron) or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μmor less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less,500 μm or less, 800 μm or less, 1 mm (millimeter) or less, 2 mm or less,3 mm or less, 5 mm or less, 10 mm or less, or in a range between any twoof these values.

The device, apparatus, or method of any prior claim, wherein the sampleholder comprises a first plate and a second plate, wherein each of theplate has a thickness of 500 nm or less, 1000 nm or less, 2 μm (micron)or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μm or less, 100μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm orless, 800 μm or less, 1 mm (millimeter) or less, 2 mm or less, 3 mm orless, 5 mm or less, 10 mm or less, or in a range between any two ofthese values.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 500 um or less, 400 um or less, 300 um or less, 200 um orless, 175 um or less, 150 um or less, 125 um or less, 100 um or less, 75um or less, 50 um or less, 40 um or less, 30 um or less, 20 um or less,10 um or less, 5 um or less, 4 um or less, 3 um or less, 2 um or less,1.8 um or less, 1.5 um or less, 1 um or less, 0.5 um or less, 0.2 um orless, 0.1 um or less, 50 nm or less, 20 nm or less, 10 nm or less, or ina range between any of the two values.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer, and wherein for aspecific part of the sample that has an average thickness of 500 um orless, 400 um or less, 300 um or less, 200 um or less, 175 um or less,150 um or less, 125 um or less, 100 um or less, 75 um or less, 50 um orless, 40 um or less, 30 um or less, 20 um or less, 10 um or less, 5 umor less, 4 um or less, 3 um or less, 2 um or less, 1.8 um or less, 1.5um or less, 1 um or less, 0.5 um or less, 0.2 um or less, 0.1 um orless, 50 nm or less, 20 nm or less, 10 nm or less, or in a range betweenany of the two values, only the interference rich regions exist.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer, wherein for aspecific part of the sample that has an average thickness of 500 um orless, 400 um or less, 300 um or less, 200 um or less, 175 um or less,150 um or less, 125 um or less, 100 um or less, 75 um or less, 50 um orless, 40 um or less, 30 um or less, 20 um or less, 10 um or less, 5 umor less, 4 um or less, 3 um or less, 2 um or less, 1.8 um or less, 1.5um or less, 1 um or less, 0.5 um or less, 0.2 um or less, 0.1 um orless, 50 nm or less, 20 nm or less, 10 nm or less, or in a range betweenany of the two values, only the interference poor regions exist.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of in a range of 0.5-2 um, 0.5-3 um, 0.5-5 um, 0.5-10 um,0.5-20 um, 0.5-30 um, or 0.5-50 um.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 500 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 200 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 100 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 50 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 25 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 10 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 5 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 3 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 2 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 1 um or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 500 nm or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness of 100 nm or less.

The device, apparatus, or method of any prior claim, wherein at leastpart of the sample is compressed into a thin layer that has an averagethickness in the range of 0.5-2 um, 0.5-3 um, or 0.5-5 um.

The device, apparatus, or method of any prior claim, wherein the averagethickness of the layer of uniform thickness is in the range of 2 um to2.2 um and the sample is blood.

The device, apparatus, or method of any prior claim, wherein the averagethickness of the layer of uniform thickness is in the range of 2.2 um to2.6 um and the sample is blood.

The device, apparatus, or method of any prior claim, wherein the averagethickness of the layer of uniform thickness is in the range of 1.8 um to2 um and the sample is blood.

The device, apparatus, or method of any prior claim, wherein the averagethickness of the layer of uniform thickness is in the range of 2.6 um to3.8 um and the sample is blood.

The device, apparatus, or method of any prior claim, wherein the averagethickness of the layer of uniform thickness is in the range of 1.8 um to3.8 um and the sample is whole blood without a dilution by anotherliquid.

The device, apparatus, or method of any prior claim, wherein the averagethickness of the layer of uniform thickness is about equal to a minimumdimension of an analyte in the sample.

The device, apparatus, or method of any prior claim, wherein the finalsample thickness device is configured to analyze the sample in 300seconds or less.

The device, apparatus, or method of any prior claim, wherein the finalsample thickness device is configured to analyze the sample in 180seconds or less.

The device, apparatus, or method of any prior claim, wherein the finalsample thickness device is configured to analyze the sample in 60seconds or less.

The device, apparatus, or method of any prior claim, wherein the finalsample thickness device is configured to analyze the sample in 30seconds or less.

The device, apparatus, or method of any prior claim, wherein the imagercomprises a camera.

The device, apparatus, or method of any prior claim, wherein the imageris a part of the detector.

The device, apparatus, or method of any prior claim, wherein the imageris the entirety of the detector.

The device, apparatus, or method of any prior claim, wherein the imageris directed by the software to capture one or more images of the sample,identify the interference element regions and the interference elementfree regions, and digitally separate the interference element regionsfrom the interference element free regions.

The device, apparatus, or method of any prior claim, wherein the imagercomprises a filter that is configured to filter signals from the sample.

The device, apparatus, or method of any prior claim, wherein the imagercomprises a light source that is configured to illuminate the sample.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for detection of proteins, peptides,nucleic acids, synthetic compounds, inorganic compounds, organiccompounds, bacteria, virus, cells, tissues, nanoparticles, and othermolecules, compounds, mixtures and substances thereof.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for diagnostics, management, and/orprevention of human diseases and conditions.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for diagnostics, management, and/orprevention of veterinary diseases and conditions, or for diagnostics,management, and/or prevention of plant diseases and conditions.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for environments testing anddecontamination.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for agricultural or veterinaryapplications.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for food testing.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for drug testing and prevention.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for detecting and/or measuring an analytein blood.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for a colorimetric assay.

The device, apparatus, or method of any prior claim, wherein theapparatus or method are used for a fluorescence assay.

The device, apparatus, or method of any prior claim, wherein the platesare movable relative to each.

The device, apparatus, or method of any prior claim, wherein the spacersare fixed on one or both of the plates and have a uniform height.

The device, apparatus, or method of any prior claim, wherein the firstplate and second plate are configured to compress the sample into alayer of uniform thickness that substantially equals the height of thespacers.

The device, apparatus, or method of any prior claim, wherein the spacershave a uniform height of 1 mm or less, 500 um or less, 400 um or less,300 um or less, 200 um or less, 175 um or less, 150 um or less, 125 umor less, 100 um or less, 75 um or less, 50 um or less, 40 um or less, 30um or less, 20 um or less, 10 um or less, 5 um or less, 4 um or less, 3um or less, 2 um or less, 1.8 um or less, 1.5 um or less, 1 um or less,0.5 um or less, 0.2 um or less, 0.1 um or less, 50 nm or less, 20 nm orless, 10 nm or less, or in a range between any of the two values.

The device, apparatus, or method of any prior claim, wherein the spacershave a uniform height in the range of 0.5-2 um, 0.5-3 um, 0.5-5 um,0.5-10 um, 0.5-20 um, 0.5-30 um, or 0.5-50 um.

The device, apparatus, or method of any prior claim, wherein at leastone of the plates has a thickness of 100 mm or less, 50 mm or less, 25mm or less, 10 mm or less, 5 mm or less, 1 mm or less, 500 um or less,400 um or less, 300 um or less, 200 um or less, 175 um or less, 150 umor less, 125 um or less, 100 um or less, 75 um or less, 50 um or less,40 um or less, 30 um or less, 20 um or less, 10 um or less, 5 um orless, 4 um or less, 3 um or less, 2 um or less, 1.8 um or less, 1.5 umor less, 1 um or less, 0.5 um or less, 0.2 um or less, or 0.1 um orless, or in a range between any of the two values.

The device, apparatus, or method of any prior claim, wherein at leastone of the plates has a thickness in the range of 0.5 to 1.5 mm; around1 mm; in the range of 0.15 to 0.2 mm; or around 0.175 mm.

The device, apparatus, or method of any prior claim, wherein at leastone of the plates has a lateral area of 1 mm² or less, 10 mm² or less,25 mm² or less, 50 mm² or less, 75 mm² or less, 1 cm² (squarecentimeter) or less, 2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm²or less, 10 cm² or less, 100 cm² or less, 500 cm² or less, 1000 cm² orless, 5000 cm² or less, 10,000 cm² or less, 10,000 cm² or less, or in arange between any two of these values

The device, apparatus, or method of any prior claim, wherein at leastone of the plates has a lateral area of in the range of 500 to 1000 mm²;or around 750 mm²

The device, apparatus, or method of any prior claim, wherein the Young'smodulus of the spacers times the filling factor of the spacers is equalor larger than 10 MPa, wherein the filling factor is the ratio of thespacer area in contact with the layer of uniform thickness to the totalplate area in contact with the layer of uniform thickness.

The device, apparatus, or method of any prior claim, wherein thethickness of the flexible plate times the Young's modulus of theflexible plate is in the range 60 to 750 GPa-urn.

The device, apparatus, or method of any prior claim, wherein for aflexible plate, the fourth power of the inter-spacer-distance (ISD)divided by the thickness of the flexible plate (h) and the Young'smodulus (E) of the flexible plate, ISD⁴/(hE), is equal to or less than10⁶ um³/GPa.

The device, apparatus, or method of any prior claim, wherein one or bothplates comprises a location marker, either on a surface of or inside theplate, that provide information of a location of the plate.

The device, apparatus, or method of any prior claim, wherein one or bothplates comprises a scale marker, either on a surface of or inside theplate, that provide information of a lateral dimension of a structure ofthe sample and/or the plate.

The device, apparatus, or method of any prior claim, wherein one or bothplates comprises an image marker, either on a surface of or inside theplate, that assists an imaging of the sample.

The device, apparatus, or method of any prior claim, wherein theinter-spacer distance is in the range of 7 um to 120 um.

The device, apparatus, or method of any prior claim, wherein theinter-spacer distance is in the range of 120 um to 200 um.

The device, apparatus, or method of any prior claim, wherein the spacersare pillars with a cross-sectional shape selected from round, polygonal,circular, square, rectangular, oval, elliptical, or any combination ofthe same.

The device, apparatus, or method of any prior claim, wherein the spacershave a pillar shape and have a substantially flat top surface, wherein,for each spacer, the ratio of the lateral dimension of the spacer to itsheight is at least 1.

The device, apparatus, or method of any prior claim, wherein each spacerhas the ratio of the lateral dimension of the spacer to its height is atleast 1.

The device, apparatus, or method of any prior claim, wherein the minimumlateral dimension of spacer is less than or substantially equal to theminimum dimension of an analyte in the sample.

The device, apparatus, or method of any prior claim, wherein the minimumlateral dimension of spacer is in the range of 0.5 um to 100 um.

The device, apparatus, or method of any prior claim, wherein the minimumlateral dimension of spacer is in the range of 0.5 um to 10 um.

The device, apparatus, or method of any prior claim, wherein the spacershave a pillar shape, and the sidewall corners of the spacers have around shape with a radius of curvature at least 1 μm.

The device, apparatus, or method of any prior claim, wherein the spacershave a density of at least 100/mm².

The device, apparatus, or method of any prior claim, wherein the spacershave a density of at least 1000/mm².

The device, apparatus, or method of any prior claim, wherein at leastone of the plates is transparent

The device, apparatus, or method of any prior claim, wherein at leastone of the plates is made from a flexible polymer.

The device, apparatus, or method of any prior claim, wherein, for apressure that compresses the plates, the spacers are not compressibleand/or, independently, only one of the plates is flexible.

The device, apparatus, or method of any prior claim, wherein theflexible plate has a thickness in the range of 10 um to 200 um.

The device, apparatus, or method of any prior claim, wherein thevariation of sample thickness is less than 30%.

The device, apparatus, or method of any prior claim, wherein thevariation of sample thickness is less than 10%.

The device, apparatus, or method of any prior claim, wherein thevariation of sample thickness is less than 5%.

The device, apparatus, or method of any prior claim, wherein the firstand second plates are connected and are configured to be changed fromthe open configuration to the closed configuration by folding theplates.

The device, apparatus, or method of any prior claim, wherein the firstand second plates are connected by a hinge and are configured to bechanged from the open configuration to the closed configuration byfolding the plates along the hinge.

The device, apparatus, or method of any prior claim, wherein the firstand second plates are connected by a hinge that is a separate materialto the plates, and are configured to be changed from the openconfiguration to the closed configuration by folding the plates alongthe hinge.

The device, apparatus, or method of any prior claim, wherein the firstand second plates are made in a single piece of material and areconfigured to be changed from the open configuration to the closedconfiguration by folding the plates.

The device, apparatus, or method of any prior claim, wherein the layerof uniform thickness sample is uniform over a lateral area that is atleast 1 mm².

The device, apparatus, or method of any prior claim, wherein the spacersare fixed on a plate by directly embossing the plate or injectionmolding of the plate.

The device, apparatus, or method of any prior claim, wherein thematerials of the plate and the spacers are selected from polystyrene,PMMA, PC, COC, COP, or another plastic.

The device, method, or system of any prior claim, wherein the firstplate further comprises a scattering surface on an inner surfacethereof.

The device, method, or system of any prior claim, wherein the firstplate is substantially transparent.

The device, method, or system of any prior claim, wherein the firstplate has a reflectivity in a range from 1% to 80%.

The device, method, or system of any prior claim, wherein the firstplate has a reflectivity in a range that is larger than 1%, 2%, 4%, 8%,10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%, or in a range between any ofthe two values.

The device, method, or system of any prior claim, wherein the firstplate is made from PMMA.

The device, method, or system of any prior claim, wherein the secondplate has a reflectivity in a range from 1% to 100%.

The device, method, or system of any prior claim, wherein the secondplate has a reflectivity in a range that is larger than 1%, 2%, 4%, 8%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or in a rangebetween any of the two values.

The device, method, or system of any prior claim, wherein at least apart of the inner surface and the outer surface of the first plate issubstantially flat.

The device, method, or system of any prior claim, wherein the scatteringsurface has a reflectivity larger than 90% over the entire spectrum ofthe illuminating light for illuminating the sample.

The device, method, or system of any prior claim, wherein the scatteringsurface has a reflectivity larger than 50%, 60%, 70%, 80%, 90%, or in arange between any of the two values.

The device, method, or system of any prior claim, wherein the scatteringsurface is coated with a metal film.

The device, method, or system of any prior claim, wherein the scatteringsurface is coated with a metal film and the metal film has a thicknessthereof in a range from 10 nm to 100 nm.

The device, method, or system of any prior claim, wherein the scatteringsurface is coated with a metal film and the thickness of the metal filmis less than 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 80nm, or 100 nm, or in a range between any of the two values.

The device, method, or system of any prior claim, wherein the scatteringsurface is coated with a metal film and the metal film includes one ormore of aluminum film, silver film, and gold film.

The device, method, or system of any prior claim, the scattering surfacehas a transmissibility in a range from 10% to 30%.

The device, method, or system of any prior claim, the scattering surfacehas a transmissibility that is less than 10%, 15%, 20%, 25%, or 30%, orin a range between any of the two values.

The device, method, or system of any prior claim, wherein the one plateof the first and second plates and the scattering surface of the otherplate enhances the light trapping.

The device, method, or system of any prior claim, wherein the scatteringsurface has a reflectance that is dominated by Lambertian reflectance inthe at least one wavelength range.

The device of, method, or system of any prior claim, wherein thescattering surface has a Lambertian reflectance that is larger than 0.8in the at least one wavelength range.

The device, method, or system of any prior claim, wherein the scatteringsurface is a surface formed by chemical etching.

The device, method, or system of any prior claim, wherein the scatteringsurface is a surface formed by nanoimprint lithography.

The device, method, or system of any prior claim, wherein the scatteringsurface covers substantially all of the sample contact area of the firstplate.

The device, method, or system of any prior claim, wherein the scatteringsurface covers a fraction of the sample contact area of the first plate.

The device, method, or system of any prior claim, wherein the part ofthe first plate is substantially transparent covers substantially all ofthe sample contact area of the second plate.

The device, method, or system of any prior claim, wherein the part ofthe first plate is substantially transparent covers a fraction of thesample contact area of the second plate.

The device, method, or system of any prior claim, wherein the scatteringsurface comprises a bumpy and wavy roughly surface.

The device, method, or system of any prior claim, wherein the scatteringsurface comprises a periodic texture.

The device, method, or system of any prior claim, wherein the scatteringsurface comprises an aperiodic texture.

The device, method, or system of any prior claim, wherein the scatteringsurface has an average roughness in a range between 2 μm to 5 μm.

The device, method, or system of any prior claim, wherein the scatteringsurface has an average roughness that is less than 2.0 μm, 2.5 μm, 3.0μm, 3.5 μm, 4.0 μm, 4.5 μm, or 5.0 μm, or in a range between any of thetwo values.

The device, method, or system of any prior claim, wherein thelight-guiding spacers are fixed to the inner surface of the first plateand have a predetermined uniform height.

The device, method, or system of any prior claim, wherein the adaptercomprises:

-   -   an adaptor housing that has an exit aperture for positioning an        imager;    -   a passive illuminator; and    -   wherein the passive illuminator is on the adaptor and is        positioned around the outside peripheral of exit aperture.

The device, method, or system of any prior claim, wherein the adaptorhousing is configured to reduce ambient light outside the adaptorhousing entering into inside adaptor housing.

The device, method, or system of any prior claim, wherein the adaptorcomprises one or two light-guides each having an end thereof alignedwith the entrance aperture of the optics chamber to cause light enteringsuch end of the light-guide to travel through the light-guide to reach acorresponding end of the passive illuminator.

The device, apparatus, or method of any prior claim, wherein one or morelight-guides and the passive illuminator are jointly formed by anoptical fiber.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is in the form of ring configured to surround an opticalaxis of a lens in the camera of the smartphone when the apparatus isengaged with the smartphone.

The device, apparatus, or method of any prior claim, wherein theapparatus further comprises an auxiliary lens having an optical axisthereof aligned with the optical axis of the lens in the camera of thesmartphone when the apparatus is engaged with the smartphone.

The device, apparatus, or method of any prior claim, wherein theapparatus further comprises an auxiliary lens having a diameter that isat least 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40mm, or 50 mm, or in a range between any of the two values.

The device, apparatus, or method of any prior claim, wherein theapparatus further comprises an optical condenser configured to be placedin front of the light source of the smartphone when the apparatus isengaged with the smartphone.

The device, apparatus, or method of any prior claim, wherein thediffuser comprises a volume diffusive material.

The device, apparatus, or method of any prior claim, wherein thediffuser comprises at least one textured surface.

The device, apparatus, or method of any prior claim, wherein thediffuser comprises a diffusive plate that is substantially uniform inthickness.

The device, apparatus, or method of any prior claim, wherein thediffuser comprises a diffusive plate including an area that hasthickness that is larger than an average thickness of the diffusiveplate.

The device, apparatus, or method of any prior claim, further comprisinga reflector configured to reflect light emitted from the passiveilluminator towards the diffuser.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is formed by a side illumination fiber.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is rotationally symmetric.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is in the form of a circle.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is in the form of a circle having a diameter thereof in arange between 5 mm and 100 mm.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is in the form of a circle having a diameter that is atleast 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 60 mm, 80mm, or 100 mm, or in a range between any of the two values.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is in the form of a convex polygon.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is in the form of a star polygon.

The device, apparatus, or method of any prior claim, wherein the opticalaxis of the lens passes through a center of the passive illuminator.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is rotationally non-symmetric.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator is in the form of an ellipse.

The device, apparatus, or method of any prior claim, wherein the passiveilluminator has a substantially uniform cross-section.

The device, apparatus, or method of any prior claim, wherein all of thecross-sections at locations on more than 50% length of the passiveilluminator are substantially identical in shape.

The device, apparatus, or method of any prior claim, wherein the shapesof substantially all of the cross-sections are in the form of a circle.

The device, apparatus, or method of any prior claim, wherein the shapesof substantially all the cross-sections are in the form of a circlehaving a diameter thereof in a range between 1.0 mm and 3.0 mm.

The device, apparatus, or method of any prior claim, wherein the shapesof substantially all of the cross-sections are in the form of a circlehaving a diameter that is at least 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5mm, 3 mm, 4 mm, 5 mm, 6 mm, 8 mm, or 10 mm, or in a range between any ofthe two values.

The device, apparatus, or method of any prior claim, wherein the shapesof substantially all of the cross-sections are in the form of anellipse.

The device, apparatus, or method of any prior claim, wherein at least asegment of the side wall of the passive illuminator is formed by adiffusive surface.

The device, method, or system of any prior claim, wherein the sample isoriginal, diluted, or processed forms of: bodily fluids, stool, amnioticfluid, aqueous humour, vitreous humour, blood, whole blood, fractionatedblood, plasma, serum, breast milk, cerebrospinal fluid, cerumen, chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus, nasal drainage, phlegm, pericardial fluid, peritoneal fluid,pleural fluid, pus, rheum, saliva, sebum, semen, sputum, sweat, synovialfluid, tears, vomit, urine, or exhaled breath condensate.

The device, method, or system of any prior claim, wherein the sample isoriginal, diluted, or processed forms of blood.

The device, method, or system of any prior claim, wherein the samplecomprises whole blood.

The device, method, or system of any prior claim, wherein the sample isa biological sample, a chemical sample, an environmental sample, or afoodstuff sample.

The device, method, or system of any prior claim, wherein the analyte isa biomarker, an environmental marker, or a foodstuff marker.

The device, method, or system of any prior claim, wherein the analyte isa biomarker indicative of the presence or severity of a disease orcondition.

The device, method, or system of any prior claim, wherein the analyte isa cell, a protein, or a nucleic acid.

The device, method, or system of any prior claim, wherein the analytecomprises proteins, peptides, nucleic acids, synthetic compounds,inorganic compounds, organic compounds, bacteria, virus, cells, tissues,nanoparticles, and other molecules, compounds, mixtures and substancesthereof.

The device, method, or system of any prior claim, wherein the sampleholder comprises wells that configured to hold the sample.

The device, method, or system of any prior claim, wherein the sampleholder comprises a first plate, and a second plate, and spacers.

The device, method, or system of any prior claim, wherein the sampleholder comprises a first plate, a second plate, and spacers, wherein thespacers are configured to regulate a gap between the plates when theplates are pressed against each, compressing the sample into a thinlayer.

The device, method, or system of any prior claim, wherein the sampleholder comprises a first plate, a second plate, and spacers, andwherein:

-   -   the plates are moveable relative to each other into different        configurations, including an open configuration and a closed        configuration;    -   in the open configuration: the two plates are separated apart,        the spacing between the plates is not regulated by the spacers,        and the sample is deposited on one or both of the plates; and    -   in the closed configuration, which is configured after the        sample deposition in the open configuration: at least part of        the sample is compressed by the two plates into a layer of        highly uniform thickness and is substantially stagnant relative        to the plates, wherein the uniform thickness of the layer is        regulated by the plates and the spacers.

The device, method, or system of any prior claim, wherein the sampleholder comprises a Q-card, which comprises a first plate, a secondplate, and spacers, wherein the spacers are configured to regulate a gapbetween the plates when the plates are pressed against each, compressingthe sample into a thin layer.

The device, method, or system of any prior claim, wherein

-   -   the sample holder comprises a first plate, a second plate, and        spacers, wherein the spacers have a uniform height and a        constant inter-spacer distance; and    -   the sample is compressed by the sample holder into a thin layer        with a uniform thickness that is regulated by the height of the        spacers.

The device, method, or system of any prior claim, wherein the sample iscompressed into a layer of uniform thickness that substantially equalsuniform height of spacers that are fixed to one or both of the plates.

The device, method, or system of any prior claim, wherein the sample iscompressed into a layer of uniform thickness that has a variation ofless than 15%, 10%, 5%, 2%, 1%, or in a range between any of the twovalues.

The device, method, or system of any prior claim, wherein in the closedconfiguration, the sample has a thickness of 500 nm or less, 1000 nm orless, 2 μm (micron) or less, 5 μm or less, 10 μm or less, 20 μm or less,50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm orless, 500 μm or less, 800 μm or less, 1 mm (millimeter) or less, 2 mm orless, 3 mm or less, 5 mm or less, 10 mm or less, or in a range betweenany two of these values.

The device, method, or system of any prior claim, wherein in the closedconfiguration, the sample has a thickness in the range of 0.5-20 μm.

The device, method, or system of any prior claim, wherein in the closedconfiguration, a gap between the first plate and the second plate is 500nm or less, 1000 nm or less, 2 μm (micron) or less, 5 μm or less, 10 μmor less, 20 μm or less, 50 μm or less, 100 μm or less, 150 μm or less,200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm(millimeter) or less, 2 mm or less, 3 mm or less, 5 mm or less, 10 mm orless, or in a range between any two of these values.

The device, method, or system of any prior claim, wherein the sampleholder comprises a first plate and a second plate, wherein each of theplate has a thickness of 500 nm or less, 1000 nm or less, 2 μm (micron)or less, 5 μm or less, 10 μm or less, 20 μm or less, 50 μm or less, 100μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm orless, 800 μm or less, 1 mm (millimeter) or less, 2 mm or less, 3 mm orless, 5 mm or less, 10 mm or less, or in a range between any two ofthese values.

The apparatus, kit, or method of any prior claim, wherein the imagercomprises a camera.

The apparatus, kit, or method of any prior claim, wherein the imager isa part of the detector.

The apparatus, kit, or method of any prior claim, wherein the imager isthe entirety of the detector.

The apparatus, kit, or method of any prior claim, wherein the imager isdirected by the software to capture one or more images of the sample,identify the interference element regions and the interference elementfree regions, and digitally separate the interference element regionsfrom the interference element free regions.

The apparatus, kit, or method of any prior claim, wherein the imagercomprises a filter that is configured to filter signals from the sample.

The apparatus, kit, or method of any prior claim, wherein the imagercomprises a light source that is configured to illuminate the sample.

The apparatus, kit, or method of any prior claim, wherein the detectoris a mobile device.

The apparatus, kit, or method of any prior claim, wherein the detectoris a smart phone.

The apparatus, kit, or method of any prior claim, wherein the detectoris a smart phone and the imager is a camera as part of the smart phone.

The apparatus, kit, or method of any prior claim, wherein the detectorcomprises a display that is configured to show the presence and/oramount of the analyte.

The apparatus, kit, or method of any prior claim, wherein the detectoris configured to transmit detection results to a third party.

The apparatus, kit, or method of any prior claim, wherein the softwareis stored in a storage unit, which is part of the detector.

The apparatus, kit, or method of any prior claim, wherein the softwareis configured to direct the detector to display the presence and/oramount of the analyte.

The apparatus, kit, or method of any prior claim, wherein the softwareis configured to direct the imager to calculate the combined signal ofthe analyte from the interference element free regions.

The apparatus, kit, or method of any prior claim, wherein the softwareis configured to direct the imager to disregard the signal of theanalyte from the interference element regions.

The apparatus, kit, or method of any prior claim, wherein the softwareis configured to direct the imager to increase signal contrast of thesignals from the interference element regions to the signals from theinterference element free regions

The apparatus, kit, or method of any prior claim, wherein the softwareis configured to direct the detector to calculate a ratio of the signalfrom the interference element regions to the interference element freeregions.

The device, method, or system of any prior claim, wherein the mobileapparatus is a smart phone.

The device, method, or system of any prior claim, wherein the mobileapparatus comprises a set of instructions that, when executed, directthe apparatus to capture one or more images of the sample,

The device, method, or system of any prior claim, wherein the mobileapparatus comprises a light source that is configured to illuminate thesample.

The device, method, or system of any prior claim, wherein the mobileapparatus comprises a display that is configured to show the presenceand/or amount of the analyte.

The device, method, or system of any prior claim, wherein the mobileapparatus comprises a set of instructions that, when executed, directthe detector to display the presence and/or amount of the analyte.

The device, method, or system of any prior claim, wherein the mobileapparatus is configured to transmit detection results to a third party.

The device, method, or system of any prior claim, wherein the adaptorcomprises a filter that is configured to filter signals from the sample.

The device, method, or system of any prior claim, wherein the adaptorcomprises a card slot, into which the device can be inserted.

The device, method, or system of any prior claim, wherein the adaptorcomprises a slider that facilitates the insertion of the device into thecard slot.

The device, method, or system of any prior claim, wherein the adaptorcomprises a holder frame that is configured to removably connect to themobile apparatus.

The device, method, or system of any prior claim, wherein the adaptorcomprises an optical box that comprises one or more optical componentsthat are configured to enhance the signal from the sample.

The device, method or system of any prior claim, wherein the apparatusor method are used for detection of proteins, peptides, nucleic acids,synthetic compounds, inorganic compounds, organic compounds, bacteria,virus, cells, tissues, nanoparticles, and other molecules, compounds,mixtures and substances thereof.

The device, method or system of any prior claim, wherein the apparatusor method are used for diagnostics, management, and/or prevention ofhuman diseases and conditions.

The device, method or system of any prior claim, wherein the apparatusor method are used for diagnostics, management, and/or prevention ofveterinary diseases and conditions, or for diagnostics, management,and/or prevention of plant diseases and conditions.

The device, method or system of any prior claim, wherein the apparatusor method are used for environments testing and decontamination.

The device, method or system of any prior claim, wherein the apparatusor method are used for agricultural or veterinary applications.

The device, method or system of any prior claim, wherein the apparatusor method are used for food testing.

The device, method or system of any prior claim, wherein the apparatusor method are used for drug testing and prevention.

The device, method or system of any prior claim, wherein the apparatusor method are used for detecting and/or measuring an analyte in blood.

The device, method or system of any prior claim, wherein the apparatusor method are used for a colorimetric assay.

The device, method or system of any prior claim, wherein the apparatusor method are used for a fluorescence assay.

The device, method or system of any prior claim, wherein the signalrelated to the analyte is an electrical signal or an optical signal.

The device, method or system of any prior claim, wherein the signalrelated to the analyte is an optical signal that allows the imager tocapture images of the interference element rich region and theinterference element poor region.

The device, method or system of any prior claim, wherein the signalrelated to the analyte is from a colorimetric reaction.

The device, method or system of any prior claim, wherein the signalrelated to the analyte is produced by illuminating the sample with anillumination source.

The device, method or system of any prior claim, wherein the plates aremovable relative to each.

The device, method or system of any prior claim, wherein the spacers arefixed on one or both of the plates and have a uniform height.

The device, method or system of any prior claim, wherein the first plateand second plate are configured to compress the sample into a layer ofuniform thickness that substantially equals the height of the spacers.

The device, method or system of any prior claim, wherein the spacershave a uniform height of 1 mm or less, 500 um or less, 400 um or less,300 um or less, 200 um or less, 175 um or less, 150 um or less, 125 umor less, 100 um or less, 75 um or less, 50 um or less, 40 um or less, 30um or less, 20 um or less, 10 um or less, 5 um or less, 4 um or less, 3um or less, 2 um or less, 1.8 um or less, 1.5 um or less, 1 um or less,0.5 um or less, 0.2 um or less, 0.1 um or less, 50 nm or less, 20 nm orless, 10 nm or less, or in a range between any of the two values.

The device, method or system of any prior claim, wherein the spacershave a uniform height in the range of 0.5-2 um, 0.5-3 um, 0.5-5 um,0.5-10 um, 0.5-20 um, 0.5-30 um, or 0.5-50 um.

The device, method or system of any prior claim, wherein at least one ofthe plates has a thickness of 100 mm or less, 50 mm or less, 25 mm orless, 10 mm or less, 5 mm or less, 1 mm or less, 500 um or less, 400 umor less, 300 um or less, 200 um or less, 175 um or less, 150 um or less,125 um or less, 100 um or less, 75 um or less, 50 um or less, 40 um orless, 30 um or less, 20 um or less, 10 um or less, 5 um or less, 4 um orless, 3 um or less, 2 um or less, 1.8 um or less, 1.5 um or less, 1 umor less, 0.5 um or less, 0.2 um or less, or 0.1 um or less, or in arange between any of the two values.

The device, method or system of any prior claim, wherein at least one ofthe plates has a thickness in the range of 0.5 to 1.5 mm; around 1 mm;in the range of 0.15 to 0.2 mm; or around 0.175 mm.

The device, method or system of any prior claim, wherein at least one ofthe plates has a lateral area of 1 mm² or less, 10 mm² or less, 25 mm²or less, 50 mm² or less, 75 mm² or less, 1 cm² (square centimeter) orless, 2 cm² or less, 3 cm² or less, 4 cm² or less, 5 cm² or less, 10 cm²or less, 100 cm² or less, 500 cm² or less, 1000 cm² or less, 5000 cm² orless, 10,000 cm² or less, 10,000 cm² or less, or in a range between anytwo of these values

The device, method or system of any prior claim, wherein at least one ofthe plates has a lateral area of in the range of 500 to 1000 mm²; oraround 750 mm²

The device, method or system of any prior claim, wherein the Young'smodulus of the spacers times the filling factor of the spacers is equalor larger than 10 M Pa, wherein the filling factor is the ratio of thespacer area in contact with the layer of uniform thickness to the totalplate area in contact with the layer of uniform thickness.

The device, method or system of any prior claim, wherein the thicknessof the flexible plate times the Young's modulus of the flexible plate isin the range 60 to 750 GPa-um.

The device, method or system of any prior claim, wherein for a flexibleplate, the fourth power of the inter-spacer-distance (ISD) divided bythe thickness of the flexible plate (h) and the Young's modulus (E) ofthe flexible plate, ISD⁴/(hE), is equal to or less than 10⁶ um³/GPa.

The device, method or system of any prior claim, wherein one or bothplates comprises a location marker, either on a surface of or inside theplate, that provide information of a location of the plate.

The device, method or system of any prior claim, wherein one or bothplates comprises a scale marker, either on a surface of or inside theplate, that provide information of a lateral dimension of a structure ofthe sample and/or the plate.

The device, method or system of any prior claim, wherein one or bothplates comprises an image marker, either on a surface of or inside theplate, that assists an imaging of the sample.

The device, method or system of any prior claim, wherein theinter-spacer distance is in the range of 7 um to 50 um.

The device, method or system of any prior claim, wherein theinter-spacer distance is in the range of 50 um to 120 um.

The device, method or system of any prior claim, wherein theinter-spacer distance is in the range of 120 um to 200 um.

The device, method or system of any prior claim, wherein the spacers arepillars with a cross-sectional shape selected from round, polygonal,circular, square, rectangular, oval, elliptical, or any combination ofthe same.

The device, method or system of any prior claim, wherein the spacershave a pillar shape and have a substantially flat top surface, wherein,for each spacer, the ratio of the lateral dimension of the spacer to itsheight is at least 1.

The device, method or system of any prior claim, wherein each spacer hasthe ratio of the lateral dimension of the spacer to its height is atleast 1.

The device, method or system of any prior claim, wherein the minimumlateral dimension of spacer is less than or substantially equal to theminimum dimension of an analyte in the sample.

The device, method or system of any prior claim, wherein the minimumlateral dimension of spacer is in the range of 0.5 um to 100 um.

The device, method or system of any prior claim, wherein the minimumlateral dimension of spacer is in the range of 0.5 um to 10 um.

The device, method or system of any prior claim, wherein the spacershave a pillar shape, and the sidewall corners of the spacers have around shape with a radius of curvature at least 1 μm.

The device, method or system of any prior claim, wherein the spacershave a density of at least 100/mm².

The device, method or system of any prior claim, wherein the spacershave a density of at least 1000/mm².

The device, method or system of any prior claim, wherein at least one ofthe plates is transparent

The device, method or system of any prior claim, wherein at least one ofthe plates is made from a flexible polymer.

The device, method or system of any prior claim, wherein, for a pressurethat compresses the plates, the spacers are not compressible and/or,independently, only one of the plates is flexible.

The device, method or system of any prior claim, wherein the flexibleplate has a thickness in the range of 10 um to 200 um.

The device, method or system of any prior claim, wherein the variationof sample thickness is less than 30%.

The device, method or system of any prior claim, wherein the variationof sample thickness is less than 10%.

The device, method or system of any prior claim, wherein the variationof sample thickness is less than 5%.

The device, method or system of any prior claim, wherein the first andsecond plates are connected and are configured to be changed from theopen configuration to the closed configuration by folding the plates.

The device, method or system of any prior claim, wherein the first andsecond plates are connected by a hinge and are configured to be changedfrom the open configuration to the closed configuration by folding theplates along the hinge.

The device, method or system of any prior claim, wherein the first andsecond plates are connected by a hinge that is a separate material tothe plates, and are configured to be changed from the open configurationto the closed configuration by folding the plates along the hinge.

The device, method or system of any prior claim, wherein the first andsecond plates are made in a single piece of material and are configuredto be changed from the open configuration to the closed configuration byfolding the plates.

The device, method or system of any prior claim, wherein the layer ofuniform thickness sample is uniform over a lateral area that is at least1 mm².

The device, method or system of any prior claim, wherein the spacers arefixed on a plate by directly embossing the plate or injection molding ofthe plate.

The device, method or system of any prior claim, wherein the materialsof the plate and the spacers are selected from polystyrene, PMMA, PC,COC, COP, or another plastic.

(1) Definitions

The terms used in describing the devices/apparatus, systems, and methodsherein disclosed are defined in the current application, or in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

(2) Sample

The devices/apparatus, systems, and methods herein disclosed can beapplied to manipulation and detection of various types of samples. Thesamples are herein disclosed, listed, described, and/or summarized inPCT Application (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

The devices, apparatus, systems, and methods herein disclosed can beused for samples such as but not limited to diagnostic samples, clinicalsamples, environmental samples and foodstuff samples. The types ofsample include but are not limited to the samples listed, describedand/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, and are hereby incorporated byreference by their entireties.

For example, in some embodiments, the devices, apparatus, systems, andmethods herein disclosed are used for a sample that comprises cells,tissues, bodily fluids and/or a mixture thereof. In some embodiments,the sample comprises a human body fluid. In some embodiments, the samplecomprises at least one of cells, tissues, bodily fluids, stool, amnioticfluid, aqueous humour, vitreous humour, blood, whole blood, fractionatedblood, plasma, serum, breast milk, cerebrospinal fluid, cerumen, chyle,chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph,mucus, nasal drainage, phlegm, pericardial fluid, peritoneal fluid,pleural fluid, pus, rheum, saliva, sebum, semen, sputum, sweat, synovialfluid, tears, vomit, urine, and exhaled breath condensate.

In some embodiments, the devices, apparatus, systems, and methods hereindisclosed are used for an environmental sample that is obtained from anysuitable source, such as but not limited to: river, lake, pond, ocean,glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinkingwater, etc.; solid samples from soil, compost, sand, rocks, concrete,wood, brick, sewage, etc.; and gaseous samples from the air, underwaterheat vents, industrial exhaust, vehicular exhaust, etc. In certainembodiments, the environmental sample is fresh from the source; incertain embodiments, the environmental sample is processed. For example,samples that are not in liquid form are converted to liquid form beforethe subject devices, apparatus, systems, and methods are applied.

In some embodiments, the devices, apparatus, systems, and methods hereindisclosed are used for a foodstuff sample, which is suitable or has thepotential to become suitable for animal consumption, e.g., humanconsumption. In some embodiments, a foodstuff sample comprises rawingredients, cooked or processed food, plant and animal sources of food,preprocessed food as well as partially or fully processed food, etc. Incertain embodiments, samples that are not in liquid form are convertedto liquid form before the subject devices, apparatus, systems, andmethods are applied.

The subject devices, apparatus, systems, and methods can be used toanalyze any volume of the sample. Examples of the volumes include, butare not limited to, about 10 mL or less, 5 mL or less, 3 mL or less, 1microliter (μL, “uL” herein) or less, 500 μL or less, 300 μL or less,250 μL or less, 200 μL or less, 170 μL or less, 150 μL or less, 125 μLor less, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL or less, 20μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL or less, 1μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less, 0.001 μL orless, 0.0005 μL or less, 0.0001 μL or less, 10 μL or less, 1 μL or less,or a range between any two of the values.

In some embodiments, the volume of the sample includes, but is notlimited to, about 100 μL or less, 75 μL or less, 50 μL or less, 25 μL orless, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL orless, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less,0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 μL or less, 1μL or less, or a range between any two of the values. In someembodiments, the volume of the sample includes, but is not limitedto,about 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μLor less, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL orless, 0.0001 μL or less, 10 μL or less, 1 μL or less, or a range betweenany two of the values.

In some embodiments, the amount of the sample is about a drop of liquid.In certain embodiments, the amount of sample is the amount collectedfrom a pricked finger or fingerstick. In certain embodiments, the amountof sample is the amount collected from a microneedle, micropipette or avenous draw.

In certain embodiments, the sample holder is configured to hold afluidic sample. In certain embodiments, the sample holder is configuredto compress at least part of the fluidic sample into a thin layer. Incertain embodiments, the sample holder comprises structures that areconfigured to heat and/or cool the sample. In certain embodiments, theheating source provides electromagnetic waves that can be absorbed bycertain structures in the sample holder to change the temperature of thesample. In certain embodiments, the signal sensor is configured todetect and/or measure a signal from the sample. In certain embodiments,the signal sensor is configured to detect and/or measure an analyte inthe sample. In certain embodiments, the heat sink is configured toabsorb heat from the sample holder and/or the heating source. In certainembodiments, the heat sink comprises a chamber that at least partlyenclose the sample holder.

(3) Q-Card, Spacers and Uniform Sample Thickness

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards, spacers, and uniform sample thickness embodiments forsample detection, analysis, and quantification. In some embodiments, theQ-card comprises spacers, which help to render at least part of thesample into a layer of high uniformity. The structure, material,function, variation and dimension of the spacers, as well as theuniformity of the spacers and the sample layer, are herein disclosed,listed, described, and/or summarized in PCT Application (designatingU.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which wererespectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. ProvisionalApplication No. 62/456,065, which was filed on Feb. 7, 2017, U.S.Provisional Application No. 62/456,287, which was filed on Feb. 8, 2017,and U.S. Provisional Application No. 62/456,504, which was filed on Feb.8, 2017, all of which applications are incorporated herein in theirentireties for all purposes.

In using QMAX card, the two plates need to be open first for sampledeposition. However, in some embodiments, the QMAX card from a packagehas the two plates are in contact each other (e.g. a close position),and to separate them is challenges, since one or both plates are verything. To facilitate an opening of the QMAX card, opening notch ornotches are created at the edges or corners of the first plate or bothplaces, and, at the close position of the plates, a part of the secondplate placed over the opening notch, hence in the notch of the firstplate, the second plate can be lifted open without a blocking of thefirst plate.

In the QMAX assay platform, a QMAX card uses two plates to manipulatethe shape of a sample into a thin layer (e.g. by compressing). Incertain embodiments, the plate manipulation needs to change the relativeposition (termed: plate configuration) of the two plates several timesby human hands or other external forces. There is a need to design theQMAX card to make the hand operation easy and fast.

In QMAX assays, one of the plate configurations is an openconfiguration, wherein the two plates are completely or partiallyseparated (the spacing between the plates is not controlled by spacers)and a sample can be deposited. Another configuration is a closedconfiguration, wherein at least part of the sample deposited in the openconfiguration is compressed by the two plates into a layer of highlyuniform thickness, the uniform thickness of the layer is confined by theinner surfaces of the plates and is regulated by the plates and thespacers. In some embodiments, the average spacing between the two platesis more than 300 um.

In a QMAX assay operation, an operator needs to first make the twoplates to be in an open configuration ready for sample deposition, thendeposit a sample on one or both of the plates, and finally close theplates into a close position. In certain embodiments, the two plates ofa QMAX card are initially on top of each other and need to be separatedto get into an open configuration for sample deposition. When one of theplate is a thin plastic film (175 um thick PMA), such separation can bedifficult to perform by hand. The present invention intends to providethe devices and methods that make the operation of certain assays, suchas the QMAX card assay, easy and fast.

In some embodiments, the QMAX device comprises a hinge that connect twoor more plates together, so that the plates can open and close in asimilar fashion as a book. In some embodiments, the material of thehinge is such that the hinge can self-maintain the angle between theplates after adjustment. In some embodiments, the hinge is configured tomaintain the QMAX card in the closed configuration, such that the entireQMAX card can be slide in and slide out a card slot without causingaccidental separation of the two plates. In some embodiments, the QMAXdevice comprises one or more hinges that can control the rotation ofmore than two plates.

In some embodiments, the hinge is made from a metallic material that isselected from a group consisting of gold, silver, copper, aluminum,iron, tin, platinum, nickel, cobalt, alloys, or any combination ofthereof. In some embodiments, the hinge comprises a single layer, whichis made from a polymer material, such as but not limited to plastics.The polymer material is selected from the group consisting of acrylatepolymers, vinyl polymers, olefin polymers, cellulosic polymers,noncellulosic polymers, polyester polymers, Nylon, cyclic olefincopolymer (COC), poly(methyl methacrylate) (PMMB), polycarbonate (PC),cyclic olefin polymer (COP), liquid crystalline polymer (LCP), polyamide(PB), polyethylene (PE), polyimide (PI), polypropylene (PP),poly(phenylene ether) (PPE), polystyrene (PS), polyoxymethylene (POM),polyether ether ketone (PEEK), polyether sulfone (PES), poly(ethylenephthalate) (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride(PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT),fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFB),polydimethylsiloxane (PDMS), rubbers, or any combinations of thereof. Insome embodiments, the polymer material is selected from polystyrene,PMMB, PC, COC, COP, other plastic, or any combination of thereof.

In essence, the term “spacers” or “stoppers” can refer to, unless statedotherwise, the mechanical objects that set, when being placed betweentwo plates, a limit on the minimum spacing between the two plates thatcan be reached when compressing the two plates together. Namely, in thecompressing, the spacers will stop the relative movement of the twoplates to prevent the plate spacing becoming less than a preset (i.e.predetermined) value.

In some embodiments, human hands can be used to press the plates into aclosed configuration; In some embodiments, human hands can be used topress the sample into a thin layer. The manners in which hand pressingis employed are described and/or summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 filed on Aug. 10, 2016 andPCT/US0216/051775 filed on Sep. 14, 2016, and in US ProvisionalApplication Nos. 62/431,639 filed on Dec. 9, 2016, 62/456,287 filed onFeb. 8, 2017, 62/456,065 filed on Feb. 7, 2017, 62/456,504 filed on Feb.8, 2017, and 62/460,062 filed on Feb. 16, 2017, which are all herebyincorporated by reference by their entireties.

In some embodiments, human hand can be used to manipulate or handle theplates of the QMAX device. In certain embodiments, the human hand can beused to apply an imprecise force to compress the plates from an openconfiguration to a closed configuration. In certain embodiments, thehuman hand can be used to apply an imprecise force to achieve high levelof uniformity in the thickness of the sample (e.g. less than 5%, 10%,15%, or 20% variability).

(4) Hinges, Opening Notches, Recessed Edge and Sliders

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards for sample detection, analysis, and quantification. Insome embodiments, the Q-card comprises hinges, notches, recesses, andsliders, which help to facilitate the manipulation of the Q card and themeasurement of the samples. The structure, material, function, variationand dimension of the hinges, notches, recesses, and sliders are hereindisclosed, listed, described, and/or summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, whichwere respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.Provisional Application No. 62/431,639, which was filed on Dec. 9, 2016,U.S. Provisional Application No. 62/456,065, which was filed on Feb. 7,2017, U.S. Provisional Application Nos. 62/456,287 and 62/456,504, whichwas filed on Feb. 8, 2017, and U.S. Provisional Application No.62/539,660, which was filed on Aug. 1, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

In some embodiments, the QMAX device comprises opening mechanisms suchas but not limited to notches on plate edges or strips attached to theplates, making is easier for a user to manipulate the positioning of theplates, such as but not limited to separating the plates of by hand.

In some embodiments, the QMAX device comprises trenches on one or bothof the plates. In certain embodiments, the trenches limit the flow ofthe sample on the plate.

(5) Q-Card and Adaptor

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards for sample detection, analysis, and quantification. Insome embodiments, the Q-card is used together with an adaptor that isconfigured to accommodate the Q-card and connect to a mobile device sothat the sample in the Q-card can be imaged, analyzed, and/or measuredby the mobile device. The structure, material, function, variation,dimension and connection of the Q-card, the adaptor, and the mobile areherein disclosed, listed, described, and/or summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application Nos. 62/456,287 and62/456,590, which were filed on Feb. 8, 2017, U.S. ProvisionalApplication No. 62/456,504, which was filed on Feb. 8, 2017, U.S.Provisional Application No. 62/459,544, which was filed on Feb. 15,2017, and U.S. Provisional Application Nos. 62/460,075 and 62/459,920,which were filed on Feb. 16, 2017, all of which applications areincorporated herein in their entireties for all purposes.

In some embodiments, the adaptor comprises a receptacle slot, which isconfigured to accommodate the QMAX device when the device is in a closedconfiguration. In certain embodiments, the QMAX device has a sampledeposited therein and the adaptor can be connected to a mobile device(e.g. a smartphone) so that the sample can be read by the mobile device.In certain embodiments, the mobile device can detect and/or analyze asignal from the sample. In certain embodiments, the mobile device cancapture images of the sample when the sample is in the QMAX device andpositioned in the field of view (FOV) of a camera, which in certainembodiments, is part of the mobile device.

In some embodiments, the adaptor comprises optical components, which areconfigured to enhance, magnify, and/or optimize the production of thesignal from the sample. In some embodiments, the optical componentsinclude parts that are configured to enhance, magnify, and/or optimizeillumination provided to the sample. In certain embodiments, theillumination is provided by a light source that is part of the mobiledevice. In some embodiments, the optical components include parts thatare configured to enhance, magnify, and/or optimize a signal from thesample.

(6) Smartphone Detection System

The devices/apparatus, systems, and methods herein disclosed can includeor use Q-cards for sample detection, analysis, and quantification. Insome embodiments, the Q-card is used together with an adaptor that canconnect the Q-card with a smartphone detection system. In someembodiments, the smartphone comprises a camera and/or an illuminationsource The smartphone detection system, as well the associated hardwareand software are herein disclosed, listed, described, and/or summarizedin PCT Application (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application Nos. 62/456,287 and62/456,590, which were filed on Feb. 8, 2017, U.S. ProvisionalApplication No. 62/456,504, which was filed on Feb. 8, 2017, U.S.Provisional Application No. 62/459,544, which was filed on Feb. 15,2017, and U.S. Provisional Application Nos. 62/460,075 and 62/459,920,which were filed on Feb. 16, 2017, all of which applications areincorporated herein in their entireties for all purposes.

In some embodiments, the smartphone comprises a camera, which can beused to capture images or the sample when the sample is positioned inthe field of view of the camera (e.g. by an adaptor). In certainembodiments, the camera includes one set of lenses (e.g. as in iPhone™6). In certain embodiments, the camera includes at least two sets oflenses (e.g. as in iPhone™ 7). In some embodiments, the smartphonecomprises a camera, but the camera is not used for image capturing.

In some embodiments, the smartphone comprises a light source such as butnot limited to LED (light emitting diode). In certain embodiments, thelight source is used to provide illumination to the sample when thesample is positioned in the field of view of the camera (e.g. by anadaptor). In some embodiments, the light from the light source isenhanced, magnified, altered, and/or optimized by optical components ofthe adaptor.

In some embodiments, the smartphone comprises a processor that isconfigured to process the information from the sample. The smartphoneincludes software instructions that, when executed by the processor, canenhance, magnify, and/or optimize the signals (e.g. images) from thesample. The processor can include one or more hardware components, suchas a central processing unit (CPU), an application-specific integratedcircuit (ASIC), an application-specific instruction-set processor(ASIP), a graphics processing unit (GPU), a physics processing unit(PPU), a digital signal processor (DSP), a field-programmable gate array(FPGA), a programmable logic device (PLD), a controller, amicrocontroller unit, a reduced instruction-set computer (RISC), amicroprocessor, or the like, or any combination thereof.

In some embodiments, the smartphone comprises a communication unit,which is configured and/or used to transmit data and/or images relatedto the sample to another device. Merely by way of example, thecommunication unit can use a cable network, a wireline network, anoptical fiber network, a telecommunications network, an intranet, theInternet, a local area network (LAN), a wide area network (WAN), awireless local area network (WLAN), a metropolitan area network (MAN), awide area network (WAN), a public telephone switched network (PSTN), aBluetooth network, a ZigBee network, a near field communication (NFC)network, or the like, or any combination thereof.

In some embodiments, the smartphone is an iPhone™, an Android™ phone, ora Windows™ phone.

(7) Detection Methods

The devices/apparatus, systems, and methods herein disclosed can includeor be used in various types of detection methods. The detection methodsare herein disclosed, listed, described, and/or summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application Nos. 62/456,287,62/456,528, 62/456631, 62/456,522, 62/456,598, 62/456,603, and62/456,628, which were filed on Feb. 8, 2017, U.S. ProvisionalApplication Nos. 62/459,276, 62/456,904, 62/457,075, and 62/457,009,which were filed on Feb. 9, 2017, and U.S. Provisional Application Nos.62/459,303, 62/459,337, and 62/459,598, which were filed on Feb. 15,2017, and U.S. Provisional Application Nos. 62/460,083, 62/460,076,which were filed on Feb. 16, 2017, all of which applications areincorporated herein in their entireties for all purposes.

(8) Labels, Capture Agent and Detection Agent

The devices/apparatus, systems, and methods herein disclosed can employvarious types of labels, capture agents, and detection agents that areused for analytes detection. The labels are herein disclosed, listed,described, and/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

In some embodiments, the label is optically detectable, such as but notlimited to a fluorescence label. In some embodiments, the labelsinclude, but are not limited to, IRDye800CW, Alexa 790, Dylight 800,fluorescein, fluorescein isothiocyanate, succinimidyl esters ofcarboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer offluorescein dichlorotriazine, cagedcarboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine,Texas Red, propidium iodide, JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanineiodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester),tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine,green fluorescent protein, blue-shifted green fluorescent protein,cyan-shifted green fluorescent protein, red-shifted green fluorescentprotein, yellow-shifted green fluorescent protein,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives, such as acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-cacid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives:coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin120),7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriaamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2-,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives: eosin, eosin isothiocyanate, erythrosin and derivatives:erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives: 5-carboxyfluorescein(FAM),5-(4,6-dichlorotriazin-2-yl)amino- -fluorescein (DTAF),2′,7′dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl hodamine isothiocyanate (TRITC); riboflavin;5-(2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CALFluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7;IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine,coumarins and related dyes, xanthene dyes such as rhodols, resorufins,bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazidessuch as luminol, and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, fluorescent europium and terbium complexes;combinations thereof, and the like. Suitable fluorescent proteins andchromogenic proteins include, but are not limited to, a greenfluorescent protein (GFP), including, but not limited to, a GFP derivedfrom Aequoria victoria or a derivative thereof, e.g., a “humanized”derivative such as Enhanced GFP; a GFP from another species such asRenilla reniformis, Renilla mulleri, or Ptilosarcus guernyi; “humanized”recombinant GFP (hrGFP); any of a variety of fluorescent and coloredproteins from Anthozoan species; combinations thereof; and the like.

In any embodiment, the QMAX device can contain a plurality of captureagents and/or detection agents that each bind to a biomarker selectedfrom Tables B1, B2, B3 and/or B7 in U.S. Provisional Application No.62/234,538 and/or PCT Application No. PCT/US2016/054025., wherein thereading step d) includes obtaining a measure of the amount of theplurality of biomarkers in the sample, and wherein the amount of theplurality of biomarkers in the sample is diagnostic of a disease orcondition.

In any embodiment, the capture agent and/or detection agents can be anantibody epitope and the biomarker can be an antibody that binds to theantibody epitope. In some embodiments, the antibody epitope includes abiomolecule, or a fragment thereof, selected from Tables B4, B5 or B6 inU.S. Provisional Application No. 62/234,538 and/or PCT Application No.PCT/US2016/054025. In some embodiments, the antibody epitope includes anallergen, or a fragment thereof, selected from Table B5. In someembodiments, the antibody epitope includes an infectious agent-derivedbiomolecule, or a fragment thereof, selected from Table B6 in U.S.Provisional Application No. 62/234,538 and/or PCT Application No.PCT/US2016/054025.

In any embodiment, the QMAX device can contain a plurality of antibodyepitopes selected from Tables B4, B5 and/or B6 in U.S. ProvisionalApplication No. 62/234,538 and/or PCT Application No. PCT/US2016/054025,wherein the reading step d) includes obtaining a measure of the amountof a plurality of epitope-binding antibodies in the sample, and whereinthe amount of the plurality of epitope-binding antibodies in the sampleis diagnostic of a disease or condition.

(9) Analytes

The devices/apparatus, systems, and methods herein disclosed can beapplied to manipulation and detection of various types of analytes(including biomarkers). The analytes are herein disclosed, listed,described, and/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

The devices, apparatus, systems, and methods herein disclosed can beused for the detection, purification and/or quantification of variousanalytes. In some embodiments, the analytes are biomarkers thatassociated with various diseases. In some embodiments, the analytesand/or biomarkers are indicative of the presence, severity, and/or stageof the diseases. The analytes, biomarkers, and/or diseases that can bedetected and/or measured with the devices, apparatus, systems, and/ormethod of the present invention include the analytes, biomarkers, and/ordiseases listed, described and/or summarized in PCT Application(designating U.S.) Nos. PCT/US2016/045437 filed on Aug. 10, 2016, andPCT Application No. PCT/US2016/054025 filed on Sep. 27, 2016, and U.S.Provisional Application Nos. 62/234,538 filed on Sep. 29, 2015,62/233,885 filed on Sep. 28, 2015, 62/293,188 filed on Feb. 9, 2016, and62/305,123 filed on Mar. 8, 2016, which are all hereby incorporated byreference by their entireties. For example, the devices, apparatus,systems, and methods herein disclosed can be used in (a) the detection,purification and quantification of chemical compounds or biomoleculesthat correlates with the stage of certain diseases, e.g., infectious andparasitic disease, injuries, cardiovascular disease, cancer, mentaldisorders, neuropsychiatric disorders and organic diseases, e.g.,pulmonary diseases, renal diseases, (b) the detection, purification andquantification of microorganism, e.g., virus, fungus and bacteria fromenvironment, e.g., water, soil, or biological samples, e.g., tissues,bodily fluids, (c) the detection, quantification of chemical compoundsor biological samples that pose hazard to food safety or nationalsecurity, e.g. toxic waste, anthrax, (d) quantification of vitalparameters in medical or physiological monitor, e.g., glucose, bloodoxygen level, total blood count, (e) the detection and quantification ofspecific DNA or RNA from biosamples, e.g., cells, viruses, bodilyfluids, (f) the sequencing and comparing of genetic sequences in DNA inthe chromosomes and mitochondria for genome analysis or (g) to detectreaction products, e.g., during synthesis or purification ofpharmaceuticals.

In some embodiments, the analyte can be a biomarker, an environmentalmarker, or a foodstuff marker. The sample in some instances is a liquidsample, and can be a diagnostic sample (such as saliva, serum, blood,sputum, urine, sweat, lacrima, semen, or mucus); an environmental sampleobtained from a river, ocean, lake, rain, snow, sewage, sewageprocessing runoff, agricultural runoff, industrial runoff, tap water ordrinking water; or a foodstuff sample obtained from tap water, drinkingwater, prepared food, processed food or raw food.

In any embodiment, the sample can be a diagnostic sample obtained from asubject, the analyte can be a biomarker, and the measured the amount ofthe analyte in the sample can be diagnostic of a disease or a condition.

In any embodiment, the devices, apparatus, systems, and methods in thepresent invention can further include diagnosing the subject based oninformation including the measured amount of the biomarker in thesample. In some cases, the diagnosing step includes sending datacontaining the measured amount of the biomarker to a remote location andreceiving a diagnosis based on information including the measurementfrom the remote location.

In any embodiment, the biomarker can be selected from Tables B1, 2, 3 or7 as disclosed in U.S. Provisional Application Nos. 62/234,538,62/293,188, and/or 62/305,123, and/or PCT Application No.PCT/US2016/054,025, which are all incorporated in their entireties forall purposes. In some instances, the biomarker is a protein selectedfrom Tables B1, 2, or 3. In some instances, the biomarker is a nucleicacid selected from Tables B2, 3 or 7. In some instances, the biomarkeris an infectious agent-derived biomarker selected from Table B2. In someinstances, the biomarker is a microRNA (miRNA) selected from Table B7.

In any embodiment, the applying step b) can include isolating miRNA fromthe sample to generate an isolated miRNA sample, and applying theisolated miRNA sample to the disk-coupled dots-on-pillar antenna (QMAXdevice) array.

In any embodiment, the QMAX device can contain a plurality of captureagents that each bind to a biomarker selected from Tables B1, B2, B3and/or B7, wherein the reading step d) includes obtaining a measure ofthe amount of the plurality of biomarkers in the sample, and wherein theamount of the plurality of biomarkers in the sample is diagnostic of adisease or condition.

In any embodiment, the capture agent can be an antibody epitope and thebiomarker can be an antibody that binds to the antibody epitope. In someembodiments, the antibody epitope includes a biomolecule, or a fragmentthereof, selected from Tables B4, B5 or B6. In some embodiments, theantibody epitope includes an allergen, or a fragment thereof, selectedfrom Table B5. In some embodiments, the antibody epitope includes aninfectious agent-derived biomolecule, or a fragment thereof, selectedfrom Table B6.

In any embodiment, the QMAX device can contain a plurality of antibodyepitopes selected from Tables B4, B5 and/or B6, wherein the reading stepd) includes obtaining a measure of the amount of a plurality ofepitope-binding antibodies in the sample, and wherein the amount of theplurality of epitope-binding antibodies in the sample is diagnostic of adisease or condition.

In any embodiment, the sample can be an environmental sample, andwherein the analyte can be an environmental marker. In some embodiments,the environmental marker is selected from Table B8 in U.S. ProvisionalApplication No. 62/234,538 and/or PCT Application No. PCT/US2016/054025.

In any embodiment, the method can include receiving or providing areport that indicates the safety or harmfulness for a subject to beexposed to the environment from which the sample was obtained.

In any embodiment, the method can include sending data containing themeasured amount of the environmental marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to be exposed to the environment from which the sample wasobtained.

In any embodiment, the QMAX device array can include a plurality ofcapture agents that each binds to an environmental marker selected fromTable B8, and wherein the reading step d) can include obtaining ameasure of the amount of the plurality of environmental markers in thesample.

In any embodiment, the sample can be a foodstuff sample, wherein theanalyte can be a foodstuff marker, and wherein the amount of thefoodstuff marker in the sample can correlate with safety of thefoodstuff for consumption. In some embodiments, the foodstuff marker isselected from Table B9.

In any embodiment, the method can include receiving or providing areport that indicates the safety or harmfulness for a subject to consumethe foodstuff from which the sample is obtained.

In any embodiment, the method can include sending data containing themeasured amount of the foodstuff marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to consume the foodstuff from which the sample is obtained.

In any embodiment, the devices, apparatus, systems, and methods hereindisclosed can include a plurality of capture agents that each binds to afoodstuff marker selected from Table B9 from in U.S. ProvisionalApplication No. 62/234,538 and PCT Application No. PCT/US2016/054025,wherein the obtaining can include obtaining a measure of the amount ofthe plurality of foodstuff markers in the sample, and wherein the amountof the plurality of foodstuff marker in the sample can correlate withsafety of the foodstuff for consumption.

provided herein are kits that find use in practicing the devices,systems and methods in the present invention.

The amount of sample can be about a drop of a sample. The amount ofsample can be the amount collected from a pricked finger or fingerstick.The amount of sample can be the amount collected from a microneedle or avenous draw.

A sample can be used without further processing after obtaining it fromthe source, or can be processed, e.g., to enrich for an analyte ofinterest, remove large particulate matter, dissolve or resuspend a solidsample, etc.

Any suitable method of applying a sample to the QMAX device can beemployed. Suitable methods can include using a pipet, dropper, syringe,etc. In certain embodiments, when the QMAX device is located on asupport in a dipstick format, as described below, the sample can beapplied to the QMAX device by dipping a sample-receiving area of thedipstick into the sample.

A sample can be collected at one time, or at a plurality of times.Samples collected over time can be aggregated and/or processed (byapplying to a QMAX device and obtaining a measurement of the amount ofanalyte in the sample, as described herein) individually. In someinstances, measurements obtained over time can be aggregated and can beuseful for longitudinal analysis over time to facilitate screening,diagnosis, treatment, and/or disease prevention.

Washing the QMAX device to remove unbound sample components can be donein any convenient manner, as described above. In certain embodiments,the surface of the QMAX device is washed using binding buffer to removeunbound sample components.

Detectable labeling of the analyte can be done by any convenient method.The analyte can be labeled directly or indirectly. In direct labeling,the analyte in the sample is labeled before the sample is applied to theQMAX device. In indirect labeling, an unlabeled analyte in a sample islabeled after the sample is applied to the QMAX device to capture theunlabeled analyte, as described below.

(10) Applications

The devices/apparatus, systems, and methods herein disclosed can be usedfor various applications (fields and samples). The applications areherein disclosed, listed, described, and/or summarized in PCTApplication (designating U.S.) Nos. PCT/US2016/045437 andPCT/US0216/051775, which were respectively filed on Aug. 10, 2016 andSep. 14, 2016, U.S. Provisional Application No. 62/456,065, which wasfiled on Feb. 7, 2017, U.S. Provisional Application No. 62/456,287,which was filed on Feb. 8, 2017, and U.S. Provisional Application No.62/456,504, which was filed on Feb. 8, 2017, all of which applicationsare incorporated herein in their entireties for all purposes.

In some embodiments, the devices, apparatus, systems, and methods hereindisclosed are used in a variety of different application in variousfield, wherein determination of the presence or absence, quantification,and/or amplification of one or more analytes in a sample are desired.For example, in certain embodiments the subject devices, apparatus,systems, and methods are used in the detection of proteins, peptides,nucleic acids, synthetic compounds, inorganic compounds, organiccompounds, bacteria, virus, cells, tissues, nanoparticles, and othermolecules, compounds, mixtures and substances thereof. The variousfields in which the subject devices, apparatus, systems, and methods canbe used include, but are not limited to: diagnostics, management, and/orprevention of human diseases and conditions, diagnostics, management,and/or prevention of veterinary diseases and conditions, diagnostics,management, and/or prevention of plant diseases and conditions,agricultural uses, veterinary uses, food testing, environments testingand decontamination, drug testing and prevention, and others.

The applications of the present invention include, but are not limitedto: (a) the detection, purification, quantification, and/oramplification of chemical compounds or biomolecules that correlates withcertain diseases, or certain stages of the diseases, e.g., infectiousand parasitic disease, injuries, cardiovascular disease, cancer, mentaldisorders, neuropsychiatric disorders and organic diseases, e.g.,pulmonary diseases, renal diseases, (b) the detection, purification,quantification, and/or amplification of cells and/or microorganism,e.g., virus, fungus and bacteria from the environment, e.g., water,soil, or biological samples, e.g., tissues, bodily fluids, (c) thedetection, quantification of chemical compounds or biological samplesthat pose hazard to food safety, human health, or national security,e.g. toxic waste, anthrax, (d) the detection and quantification of vitalparameters in medical or physiological monitor, e.g., glucose, bloodoxygen level, total blood count, (e) the detection and quantification ofspecific DNA or RNA from biological samples, e.g., cells, viruses,bodily fluids, (f) the sequencing and comparing of genetic sequences inDNA in the chromosomes and mitochondria for genome analysis or (g) thedetection and quantification of reaction products, e.g., duringsynthesis or purification of pharmaceuticals.

In some embodiments, the subject devices, apparatus, systems, andmethods are used in the detection of nucleic acids, proteins, or othermolecules or compounds in a sample. In certain embodiments, the devices,apparatus, systems, and methods are used in the rapid, clinicaldetection and/or quantification of one or more, two or more, or three ormore disease biomarkers in a biological sample, e.g., as being employedin the diagnosis, prevention, and/or management of a disease conditionin a subject. In certain embodiments, the devices, apparatus, systems,and methods are used in the detection and/or quantification of one ormore, two or more, or three or more environmental markers in anenvironmental sample, e.g. sample obtained from a river, ocean, lake,rain, snow, sewage, sewage processing runoff, agricultural runoff,industrial runoff, tap water or drinking water. In certain embodiments,the devices, apparatus, systems, and methods are used in the detectionand/or quantification of one or more, two or more, or three or morefoodstuff marks from a food sample obtained from tap water, drinkingwater, prepared food, processed food or raw food.

In some embodiments, the subject device is part of a microfluidicdevice. In some embodiments, the subject devices, apparatus, systems,and methods are used to detect a fluorescence or luminescence signal. Insome embodiments, the subject devices, apparatus, systems, and methodsinclude, or are used together with, a communication device, such as butnot limited to: mobile phones, tablet computers and laptop computers. Insome embodiments, the subject devices, apparatus, systems, and methodsinclude, or are used together with, an identifier, such as but notlimited to an optical barcode, a radio frequency ID tag, or combinationsthereof.

In some embodiments, the sample is a diagnostic sample obtained from asubject, the analyte is a biomarker, and the measured amount of theanalyte in the sample is diagnostic of a disease or a condition. In someembodiments, the subject devices, systems and methods further includereceiving or providing to the subject a report that indicates themeasured amount of the biomarker and a range of measured values for thebiomarker in an individual free of or at low risk of having the diseaseor condition, wherein the measured amount of the biomarker relative tothe range of measured values is diagnostic of a disease or condition.

In some embodiments, the sample is an environmental sample, and whereinthe analyte is an environmental marker. In some embodiments, the subjectdevices, systems and methods includes receiving or providing a reportthat indicates the safety or harmfulness for a subject to be exposed tothe environment from which the sample was obtained. In some embodiments,the subject devices, systems and methods include sending data containingthe measured amount of the environmental marker to a remote location andreceiving a report that indicates the safety or harmfulness for asubject to be exposed to the environment from which the sample wasobtained.

In some embodiments, the sample is a foodstuff sample, wherein theanalyte is a foodstuff marker, and wherein the amount of the foodstuffmarker in the sample correlate with safety of the foodstuff forconsumption. In some embodiments, the subject devices, systems andmethods include receiving or providing a report that indicates thesafety or harmfulness for a subject to consume the foodstuff from whichthe sample is obtained. In some embodiments, the subject devices,systems and methods include sending data containing the measured amountof the foodstuff marker to a remote location and receiving a report thatindicates the safety or harmfulness for a subject to consume thefoodstuff from which the sample is obtained.

(11) Dimensions

The devices, apparatus, systems, and methods herein disclosed caninclude or use a QMAX device, which can comprise plates and spacers. Insome embodiments, the dimension of the individual components of the QMAXdevice and its adaptor are listed, described and/or summarized in PCTApplication (designating U.S.) No. PCT/US2016/045437 filed on Aug. 10,2016, and U.S. Provisional Application Nos. 62/431,639 filed on Dec. 9,2016 and 62/456,287 filed on Feb. 8, 2017, which are all herebyincorporated by reference by their entireties.

In some embodiments, the dimensions are listed in the Tables below:

Plates:

Para- meters Embodiments Preferred Embodiments Shape round, ellipse,rectangle, triangle, polygonal, ring- at least one of the two (orshaped, or any superposition of these shapes; the more) plates of theQMAX two (or more) plates of the QMAX card can have card has roundcorners for the same size and/or shape, or different size user safetyconcerns, and/or shape; wherein the round corners have a diameter of 100um or less, 200 um or less, 500 um or less, 1 mm or less, 2 mm or less,5 mm or less, 10 mm or less, 50 mm or less, or in a range between anytwo of the values. Thickness the average thickness for at least one ofthe plates For at least one of the is 2 nm or less, 10 nm or less, 100nm or less, plates is in the range of 0.5 200 nm or less, 500 nm orless, 1000 nm or less, to 1.5 mm; around 1 mm; 2 μm (micron) or less, 5μm or less, 10 μm or less, in the range of 0.15 to 0.2 20 μm or less, 50μm or less, 100 μm or less, mm; or around 0.175 mm 150 μm or less, 200μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm(millimeter) or less, 2 mm or less, 3 mm or less, 5 mm or less, 10 mm orless, 20 mm or less, 50 mm or less, 100 mm or less, 500 mm or less, orin a range between any two of these values Lateral For at least one ofthe plate is 1 mm2 (square For at least one plate of the Areamillimeter) or less, 10 mm2 or less, 25 mm2 or QMAX card is in the rangeless, 50 mm2 or less, 75 mm2 or less, 1 cm2 of 500 to 1000 mm²; or(square centimeter) or less, 2 cm2 or less, 3 cm2 around 750 mm². orless, 4 cm2 or less, 5 cm2 or less, 10 cm2 or less, 100 cm2 or less, 500cm2 or less, 1000 cm2 or less, 5000 cm2 or less, 10,000 cm2 or less,10,000 cm2 or less, or in a range between any two of these valuesLateral For at least one of the plates of the QMAX card is For at leastone plate of the Linear 1 mm or less, 5 mm or less, 10 mm or less, 15QMAX card is in the range Dimension mm or less, 20 mm or less, 25 mm orless, 30 of 20 to 30 mm; or around (width, mm or less, 35 mm or less, 40mm or less, 45 24 mm length, or mm or less, 50 mm or less, 100 mm orless, 200 diameter, mm or less, 500 mm or less, 1000 mm or less, etc.)5000 mm or less, or in a range between any two of these values Recess 1um or less, 10 um or less, 20 um or less, In the range of 1 mm to width30 um or less, 40 um or less, 50 um or less, 10 mm; Or 100 um or less,200 um or less, 300 um or less, About 5 mm 400 um or less, 500 um orless, 7500 um or less, 1 mm or less, 5 mm or less, 10 mm or less, 100 mmor less, or 1000 mm or less, or in a range between any two of thesevalues.

Hinge:

Parameters Embodiments Preferred Embodiments Number 1, 2, 3, 4, 5, ormore 1 or 2 Length of 1 mm or less, 2 mm or less, 3 mm or less, 4 mm Inthe range of 5 mm Hinge Joint or less, 5 mm or less, 10 mm or less, 15mm or to 30 mm. less, 20 mm or less, 25 mm or less, 30 mm or less, 40 mmor less, 50 mm or less, 100 mm or less, 200 mm or less, or 500 mm orless, or in a range between any two of these values Ratio (hinge 1.5 orless, 1 or less, 0.9 or less, 0.8 or less, 0.7 In the range of 0.2 to 1;joint length or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or orabout 1 vs. aligning less, 0.2 or less, 0.1 or less, 0.05 or less or ina plate edge range between any two of these values. length Area 1 mm² orless, 5 mm² or less, 10 mm² or less, In the range of 20 to 20 mm² orless, 30 mm² or less, 40 mm² or less, 200 mm²; or about 50 mm² or less,100 mm² or less, 200 mm² 120 mm² or less, 500 mm² or less, or in a rangebetween any of the two values Ratio (hinge 1 or less, 0.9 or less, 0.8or less, 0.7 or less, In the range of 0.05 to area vs. 0.6 or less, 0.5or less, 0.4 or less, 0.3 or less, 0.2, around 0.15 plate area) 0.2 orless, 0.1 or less, 0.05 or less, 0.01 or less or in a range between anytwo of these values Max. Open 15 or less, 30 or less, 45 or less, 60 orless, 75 or In the range of 90 to Degree less, 90 or less, 105 or less,120 or less, 135 or 180 degrees less, 150 or less, 165 or less, 180 orless, 195 or less, 210 or less, 225 or less, 240 or less, 255 or less,270 or less, 285 or less, 300 or less, 315 or less, 330 or less, 345 orless or 360 or less degrees, or in a range between any two of thesevalues No. of 1, 2, 3, 4, 5, or more 1 or 2 Layers Layer 0.1 um or less,1 um or less, 2 um or less, 3 um In the range of 20 um thickness orless, 5 um or less, 10 um or less, 20 um or to 1 mm; or less, 30 um orless, 50 um or less, 100 um Around 50 um or less, 200 um or less ,300 umor less, 500 um or less, 1 mm or less, 2 mm or less, and a range betweenany two of these values Angle- Limiting the angle adjustment with nomore than No more than ±2 maintaining ±90, ±45, ±30, ±25, ±20, ±15, ±10,±8, ±6, ±5, ±4, ±3, ±2, or ±1, or in a range between any two of thesevalues

Notch:

Parameters Embodiments Preferred Embodiments Number 1, 2, 3, 4, 5, ormore 1 or 2 Shape round, ellipse, rectangle, triangle, polygon, ring-Part of a circle shaped, or any superposition or portion of theseshapes. Positioning Any location along any edge except the hinge edge,or any corner joint by non-hinge edges Lateral 1 mm or less, 2.5 mm orless, 5 mm or less, In the range of Linear 10 mm or less, 15 mm or less,20 mm or less, 5 mm to 15 mm; Dimension 25 mm or less, 30 mm or less, 40mm or less, or about 10 mm (Length along 50 mm or less, or in a rangebetween any two the edge, of these values radius, etc.) Area 1 mm²(square millimeter) or less, 10 mm² In the range of 10 or less, 25 mm²or less, 50 mm² or less, to 150 mm²; or 75 mm² or less or in a rangebetween any about 50 mm² two of these values.

Trench:

Preferred Parameters Embodiments Embodiments Number 1, 2, 3, 4, 5, ormore 1 or 2 Shape Closed (round, ellipse, rectangle, triangle, polygon,ring-shaped, or any superposition or portion of these shapes) oropen-ended (straight line, curved line, arc, branched tree, or any othershape with open endings); Length 0.001 mm or less, 0.005 mm or less,0.01 mm or less, 0.05 mm or less, 0.1 mm or less, 0.5 mm or less, 1 mmor less, 2 mm or less, 5 mm or less, 10 mm or less, 20 mm or less, 50 mmor less, 100 mm or less, or in a range between any two of these valuesCross- 0.001 mm² or less, 0.005 mm² or less, 0.01 mm² sectional or less,0.05 mm² or less, 0.1 mm² or less, Area 0.5 mm² or less, 1 mm² or less,2 mm² or less, 5 mm² or less, 10 mm² or less, 20 mm² or less, or in arange between any two of these values. Volume 0.1 uL or more, 0.5 uL ormore, 1 uL or more, In the range of 2 uL or more, 5 uL or more, 10 uL ormore, 1 uL to 20 uL; 30 uL or more, 50 uL or more, 100 uL or or About 5uL more, 500 uL or more, 1 mL or more, or in a range between any two ofthese values

Receptacle Slot

Preferred Parameters Embodiments Embodiments Shape of round, ellipse,rectangle, triangle, polygon, ring- receiving shaped, or anysuperposition of these shapes; area Difference 100 nm, 500 nm, 1 um, 2um, 5 um, 10 um, 50 um, In the range of 50 between 100 um, 300 um, 500um, 1 mm, 2 mm, 5 mm, to 300 um; sliding track 1 cm, or in a rangebetween any two of the values. or about 75 um gap size and cardthickness Difference 1 mm² (square millimeter) or less, 10 mm² or less,between 25 mm² or less, 50 mm² or less, 75 mm² or less, receiving 1 cm²(square centimeter) or less, 2 cm² or less, area and 3 cm² or less, 4cm² or less, 5 cm² or less, 10 cm² card area or less, 100 cm² or less,or in a range between any of the two values.

(12) Cloud

The devices/apparatus, systems, and methods herein disclosed can employcloud technology for data transfer, storage, and/or analysis. Therelated cloud technologies are herein disclosed, listed, described,and/or summarized in PCT Application (designating U.S.) Nos.PCT/US2016/045437 and PCT/US0216/051775, which were respectively filedon Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional Application No.62/456,065, which was filed on Feb. 7, 2017, U.S. ProvisionalApplication No. 62/456,287, which was filed on Feb. 8, 2017, and U.S.Provisional Application No. 62/456,504, which was filed on Feb. 8, 2017,all of which applications are incorporated herein in their entiretiesfor all purposes.

In some embodiments, the cloud storage and computing technologies caninvolve a cloud database. Merely by way of example, the cloud platformcan include a private cloud, a public cloud, a hybrid cloud, a communitycloud, a distributed cloud, an inter-cloud, a multi-cloud, or the like,or any combination thereof. In some embodiments, the mobile device (e.g.smartphone) can be connected to the cloud through any type of network,including a local area network (LAN) or a wide area network (WAN).

In some embodiments, the data (e.g. images of the sample) related to thesample is sent to the cloud without processing by the mobile device andfurther analysis can be conducted remotely. In some embodiments, thedata related to the sample is processed by the mobile device and theresults are sent to the cloud. In some embodiments, both the raw dataand the results are transmitted to the cloud.

OTHER EMBODIMENTS

Further examples of inventive subject matter according to the presentdisclosure are described in the following enumerated paragraphs.

It must be noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise, e.g., when the word “single” isused. For example, reference to “an analyte” includes a single analyteand multiple analytes, reference to “a capture agent” includes a singlecapture agent and multiple capture agents, reference to “a detectionagent” includes a single detection agent and multiple detection agents,and reference to “an agent” includes a single agent and multiple agents.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. The term “about” or “approximately” can mean withinan acceptable error range for the particular value as determined by oneof ordinary skill in the art, which will depend in part on how the valueis measured or determined, e.g. the limitations of the measurementsystem. For example, “about” can mean within 1 or more than 1 standarddeviation, per the practice in the art. Alternatively, “about” can meana range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, within5-fold, and more preferably within 2-fold, of a value. Where particularvalues are described in the application and claims, unless otherwisestated the term “about” meaning within an acceptable error range for theparticular value should be assumed. The term “about” has the meaning ascommonly understood by one of ordinary skill in the art. In certainembodiments, the term “about” can refer to ±10%. In certain embodiments,the term “about” can refer to ±5%.

As used herein, the terms “adapted” and “configured” mean that theelement, component, or other subject matter is designed and/or intendedto perform a given function. Thus, the use of the terms “adapted” and“configured” should not be construed to mean that a given element,component, or other subject matter is simply “capable of” performing agiven function. Similarly, subject matter that is recited as beingconfigured to perform a particular function can additionally oralternatively be described as being operative to perform that function.

As used herein, the phrase, “for example,” the phrase, “as an example,”and/or simply the terms “example” and “exemplary” when used withreference to one or more components, features, details, structures,embodiments, and/or methods according to the present disclosure, areintended to convey that the described component, feature, detail,structure, embodiment, and/or method is an illustrative, non-exclusiveexample of components, features, details, structures, embodiments,and/or methods according to the present disclosure. Thus, the describedcomponent, feature, detail, structure, embodiment, and/or method is notintended to be limiting, required, or exclusive/exhaustive; and othercomponents, features, details, structures, embodiments, and/or methods,including structurally and/or functionally similar and/or equivalentcomponents, features, details, structures, embodiments, and/or methods,are also within the scope of the present disclosure.

As used herein, the phrases “at least one of” and “one or more of,” inreference to a list of more than one entity, means any one or more ofthe entity in the list of entity, and is not limited to at least one ofeach and every entity specifically listed within the list of entity. Forexample, “at least one of A and B” (or, equivalently, “at least one of Aor B,” or, equivalently, “at least one of A and/or B”) can refer to Aalone, B alone, or the combination of A and B.

As used herein, the term “and/or” placed between a first entity and asecond entity means one of (1) the first entity, (2) the second entity,and (3) the first entity and the second entity. Multiple entity listedwith “and/or” should be construed in the same manner, e.g., “one ormore” of the entity so conjoined. Other entity can optionally be presentother than the entity specifically identified by the “and/or” clause,whether related or unrelated to those entities specifically identified.

Where numerical ranges are mentioned herein, the invention includesembodiments in which the endpoints are included, embodiments in whichboth endpoints are excluded, and embodiments in which one endpoint isincluded and the other is excluded. It should be assumed that bothendpoints are included unless indicated otherwise. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art.

In the event that any patents, patent applications, or other referencesare incorporated by reference herein and (1) define a term in a mannerthat is inconsistent with and/or (2) are otherwise inconsistent with,either the non-incorporated portion of the present disclosure or any ofthe other incorporated references, the non-incorporated portion of thepresent disclosure shall control, and the term or incorporateddisclosure therein shall only control with respect to the reference inwhich the term is defined and/or the incorporated disclosure was presentoriginally.

What is claimed is:
 1. A device comprising: a first plate, a secondplate, a light guiding spacer (LGS), a sampling region, and a referenceregion, wherein: (i) the first plate and second plate are configured tosandwich a sample, this is for an optical transmission analysis bylight, into a thin layer between the plates, and each plate has a samplecontact area on its inner surface that contacts the sample; (ii) thelight-guiding spacer (LGS) has a pillar shape, is sandwiched between thetwo plates with each end of the light-guiding spacer (LGS) in directcontact to one of the plates forming a LGS-plate contact area, and isconfigured to allow the light to transmit from the first plate, throughthe LGS, to the second plate without going through a sample, (iii) thesampling region is the region that the light can go through, insequence, the first plate, the sample, and the second plate, wherein thesampling region does not have the LGS; and (iv) the reference region isthe region that the light transmits through, in sequence, the firstplate, the light-guiding spacer, and the second plate, without goingthrough the sample; wherein the LGS-plate contact areas and a lateralcross-section of the LGS are larger than the wavelength of the light,wherein the light-guiding spacer is surrounded by or near the sample;and wherein the sample in the sampling region has a thickness of 500 umor less.
 2. A device for analyzing hemoglobin in a sample, comprising: afirst plate, a second plate, and a light-guiding spacer, wherein: (i)the first plate and second plate are movable relative to each other intodifferent configurations, including an open configuration and a closedconfiguration; (ii) each of the plates comprises an inner surface thathas a sample contact area for contacting a sample that contains or issuspected to contain hemoglobin; and (iii) the light-guiding spacer hasa pillar shape, wherein top and bottom surfaces of the light-guidingspacers are substantially flat and the bottom surface is fixed on theinner surface of one of the plates, wherein the area of the top andbottom surfaces and average lateral cross-section of each spacer isrespectively larger than the wavelength of the light that analyzes thesample, wherein the light-guiding spacer is inside the sample contactarea; wherein the open configuration is a configuration, in which: thetwo plates are separated apart, the spacing between the plates is notregulated by the light-guiding spacers, and the sample is deposited onone or both of the plates; wherein the closed configuration is aconfiguration, which is configured after the sample deposition in theopen configuration; and in the closed configuration: at least part ofthe sample is compressed by the two plates into a layer of highlyuniform thickness, wherein the uniform thickness of the layer isconfined by the sample contact areas of the plates and is regulated bythe plates and the light-guiding spacers; and wherein in the closedconfiguration: (a) at least one spacer in the sample contact area hasits top surface in direct contact with one of the plates, and the atleast one spacer and the regions of the plates above and below the atleast one spacer define a reference region wherein the reference regionis transparent to light within a wavelength range, and (b) at least oneregion in the sample contact area on one plate and its correspondingregion on the other plate are not occupied by the light-guiding spacers,defining a sampling region that is transparent to light within the samewavelength range.
 3. A device for analyzing hemoglobin in an analyte ina sample, comprising: a first plate, a second plate, and a light-guidingspacer, wherein: (i) the first plate and second plate are configured tohold a sample that contains or is suspected to contain an analyte,wherein at least part the sample is between the two plates and is incontact with both plates; and (ii) the light-guiding spacer has a pillarshape and a predetermined substantially uniform height, wherein top andbottom surfaces of the light-guiding spacers are substantially flat andthe top and bottom surfaces of at least one spacer are in direct contactwith the plates, wherein the area of the top and bottom surfaces andaverage lateral cross-section of each spacer is respectively larger thanthe wavelength of the light that analyze the sample, and wherein: (a)the at least one spacer and the regions of the plates directly above andbelow the at least one spacer define a reference region that istransparent to light within a wavelength range and passing through theplates and the spacer, and (b) at least one region in the sample contactarea on one plate and its corresponding region on the other plate arenot occupied by the light-guiding spacers, defining a sampling regionthat is transparent to light within the same wavelength range.
 4. Adevice for analyzing an analyte in a sample, comprising: a first plate,a second plate, and light-guiding spacers, wherein: (i) the first plateand second plate are movable relative to each other into differentconfigurations, including an open configuration and a closedconfiguration; (ii) each of the plates comprises an inner surface thathas a sample contact area for contacting a sample that contains or issuspected to contain an analyte; and (iii) the light-guiding spacershave a pillar shape and a predetermined substantially uniform height,wherein top and bottom surfaces of the light-guiding spacers aresubstantially flat and the bottom surface is fixed on the inner surfaceof one or the plates, wherein the area of the top and bottom surfacesand average lateral cross-section of each spacer is respectively largerthan the wavelength of the light that analyzes the sample, wherein atleast one of the light-guiding spacers is inside the sample contactarea; wherein the open configuration is a configuration, in which: thetwo plates are separated apart, the spacing between the plates is notregulated by the light-guiding spacers, and the sample is deposited onone or both of the plates; wherein the closed configuration is aconfiguration, which is configured after the sample deposition in theopen configuration; and in the closed configuration: at least part ofthe sample is compressed by the two plates into a layer of highlyuniform thickness, wherein the uniform thickness of the layer isconfined by the sample contact areas of the plates and is regulated bythe plates and the light-guiding spacers; and wherein in the closedconfiguration, (a) at least one spacer in the sample contact area hasits top surface in direct contact with one of the plates, and the atleast one spacer and the regions of the plates above and below the atleast one spacer define a reference region wherein the reference regionis transparent to light within a wavelength range, and (b) at least oneregion in the sample contact area on one plate and its correspondingregion on the other plate are not occupied by the light-guiding spacers,defining a sampling region that is transparent to the light within thewavelength range.
 5. (canceled)
 6. An apparatus for sample analysis,comprising: a device of claim 1, a light source, a camera; and anadaptor, wherein (i) the light source is configured to emit light in thewavelength range that is configured to go through the reference region;(ii) the camera is configured to image the reference region and thesampling region. (iii) the adaptor is configured to position the device,the light source, and the camera relative to each other, so that thelight from the light source goes through the reference region and thesampling region and is imaged by the camera.
 7. The apparatus of claim6, further comprising: a processor, which is configured to process theimages captured by the camera, and determine a property of the analytein the sample based on comparing light transmissions from the referenceregion and the sampling region.
 8. The apparatus of claim 6, wherein thecamera and the processor are parts of a single mobile device.
 9. Theapparatus of claim 6, wherein the light source and the processor areparts of a single mobile device.
 10. The apparatus of claim 6, whereinthe light source, the camera, and the processor are parts of a singlemobile device.
 11. The apparatus of claim 6, wherein the mobile deviceis a smart phone.
 12. A method for sample analysis using an transmittedlight, comprising the steps of: (a) having a device of claim 1; (b)depositing the sample at the open configuration of the device, whereinthe sample is suspected of containing an analyte; (c) bringing thedevice into the closed configuration; (d) having a light source that hasa wavelength that is configured to go through the reference region ofthe device; (e) having an imager that is configured to image thereference region and the sampling region of the device; (f) having anadaptor that is configured to position the device, the light source, andthe camera relative to each other, so that the light from the lightsource goes through the reference region and the sampling region and isimaged by the camera; (g) determining a property of the analyte bycomparing the light transmission from the sampling region and thereference region.
 13. The device of claim 1, wherein the analyte ishemoglobin.
 14. The device of claim 1, wherein the analyte is type ofcells.
 15. The device of claim 1, wherein the thickness of the samplelayer is regulated by plates and the light-guiding spacers and issubstantially the same as the uniform height of the light-guidingspacers;
 16. The device of claim 1, wherein the analyte is red bloodcells.
 17. The device of claim 1, wherein the analyte is white bloodcells.
 18. The device of claim 1, wherein the reference region and thesampling region have a same size.
 19. The device of claim 1, wherein thereference region is within a corresponding area of the cross section ofthe light-guide spacer.
 20. The device of claim 1, wherein the referenceregion is less than 0.1 um{circumflex over ( )}2, less than 0.2um{circumflex over ( )}2, less than 0.5 um{circumflex over ( )}2, lessthan 1 um{circumflex over ( )}2, less than 2 um{circumflex over ( )}2,less than 5 um{circumflex over ( )}2, less than 10 um{circumflex over( )}2, less than 20 um{circumflex over ( )}2, less than 50 um{circumflexover ( )}2, less than 100 um{circumflex over ( )}2, less than 200um{circumflex over ( )}2, less than 500 um{circumflex over ( )}2, lessthan 1000 um{circumflex over ( )}2, less than 2000 um{circumflex over( )}2, less than 5000 um{circumflex over ( )}2, less than 10000um{circumflex over ( )}2, less than 20000 um{circumflex over ( )}2, lessthan 50000 um{circumflex over ( )}2, less than 100000 um{circumflex over( )}2, less than 200000 um{circumflex over ( )}2, less than 500000um{circumflex over ( )}2, less than 1 um{circumflex over ( )}2, lessthan 2 um{circumflex over ( )}2, less than 5 um{circumflex over ( )}2,less than 10 um{circumflex over ( )}2, less than 20 um{circumflex over( )}2, or less than 50 mm{circumflex over ( )}2, or in a range betweenany of the two values.
 21. The device of claim 1, wherein the devicefurther comprises a plurality of light guiding spacers that havesubstantially uniform height, and wherein at least one of thelight-guiding spacers is inside the sample contact area.
 22. The deviceof claim 1, wherein the device further comprises a plurality of lightguiding spacers that have substantially uniform height, wherein thedistance between two neighboring light guiding spacers are known, andwherein at least one of the light-guiding spacers is inside the samplecontact area.
 23. The device of claim 1, wherein the device furthercomprises a plurality of light guiding spacers that have substantiallyuniform height, wherein the distances between two neighboring lightguiding spacers are known and are substantially constant (i.e. the lightguiding spacers are substantially a periodic array), and wherein atleast one of the light-guiding spacers is inside the sample contactarea.
 24. The device of claim 1, wherein the bottom surface of the lightguiding spacer is fixed on the inner surface of one of the plates bymolding the light guiding spacer on the inner surface of the plate. 25.The device of claim 1, wherein the bottom surface of the light guidingspacer is fixed on the inner surface of one of the plates and is made ofthe same material as the inner surface.
 26. The device of claim 1,wherein the bottom surface of the light guiding spacer is fixed on theinner surface of one of the plates, and is made of the same material asthe inner surface, and the bottom surface of the light guiding spacerhas no interface with on the inner surface of the plate.
 27. The deviceof claim 1, wherein the wavelength of the light is longer than 300 nm,and wherein the wavelength of the light is also less than 20 μm, lessthan 15 μm, less than 10 μm, less than 5 μm, less than 4 μm, less than 3μm, less than 2 μm, less than 1 μm, less than 800 nm, less than 750 nm,less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm,less than 500 nm, less than 450 nm, less than 400 nm, or in a rangebetween any of the two values.
 28. The device of claim 1, wherein thewavelength of the light is longer than 500 nm, and wherein thewavelength of the light is also less than 600 nm, less than 590 nm, lessthan 580 nm, less than 570 nm, less than 560 nm, less than 550 nm, lessthan 540 nm, less than 530 nm, less than 520 nm, less than 510 nm, or ina range between any of the two values.
 29. The device of claim 1,wherein the average lateral cross-section of each light-guiding spaceris less than 1 um{circumflex over ( )}2 (micron-square), 10um{circumflex over ( )}2, 20 um{circumflex over ( )}2, 30 um{circumflexover ( )}2, 50 um{circumflex over ( )}2, 100 um{circumflex over ( )}2,150 um{circumflex over ( )}2, 200 um{circumflex over ( )}2, 300um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, 100,000 um{circumflex over ( )}2, 200,000um{circumflex over ( )}2, 500,000 um{circumflex over ( )}2, 1um{circumflex over ( )}2, 2 um{circumflex over ( )}2, 5 um{circumflexover ( )}2, 10 um{circumflex over ( )}2, 50 um{circumflex over ( )}2, orin a range between any of the two values.
 30. The device of claim 1,wherein the average lateral cross-section of each light-guiding spaceris less than 1 um{circumflex over ( )}2 (micron-square), 10um{circumflex over ( )}2, 20 um{circumflex over ( )}2, 30 um{circumflexover ( )}2, 50 um{circumflex over ( )}2, 100 um{circumflex over ( )}2,150 um{circumflex over ( )}2, 200 um{circumflex over ( )}2, 300um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, 100,000 um{circumflex over ( )}2, 200,000um{circumflex over ( )}2, or in a range between any of the two values.31. The device of claim 1, wherein the average lateral cross-section ofeach light-guiding spacer is less than 1 um{circumflex over ( )}2(micron-square), 10 um{circumflex over ( )}2, 20 um{circumflex over( )}2, 30 um{circumflex over ( )}2, 50 um{circumflex over ( )}2, 100um{circumflex over ( )}2, 150 um{circumflex over ( )}2, 200um{circumflex over ( )}2, 300 um{circumflex over ( )}2, 500um{circumflex over ( )}2, 1000 um{circumflex over ( )}2, 2000um{circumflex over ( )}2, 5000 um{circumflex over ( )}2, 10,000um{circumflex over ( )}2, 30,000 um{circumflex over ( )}2, or in a rangebetween any of the two values.
 32. The device of claim 1, wherein thesample contact area is larger than 100 um{circumflex over( )}2(micron-square), larger than 200 um{circumflex over ( )}2, largerthan 400 um{circumflex over ( )}2, larger than 600 um{circumflex over( )}2, larger than 800 um{circumflex over ( )}2, larger than 1,000um{circumflex over ( )}2, larger than 2,000 um{circumflex over ( )}2,larger than 4,000 um{circumflex over ( )}2, larger than 6,000um{circumflex over ( )}2, larger than 8,000 um{circumflex over ( )}2,larger than 10,000 um{circumflex over ( )}2, larger than 20,000um{circumflex over ( )}2, larger than 40,000 um{circumflex over ( )}2,larger than 60,000 um{circumflex over ( )}2, larger than 80,000um{circumflex over ( )}2, larger than 100,000 um{circumflex over ( )}2,larger than 200,000 um{circumflex over ( )}2, larger than 250,000um{circumflex over ( )}2, larger than 500,000 um{circumflex over ( )}2(micron-square), or in a range between any of the two values.
 33. Thedevice of claim 1, wherein a predetermined constant inter-spacerdistance that is at least about 2 times larger than the size of theanalyte.
 34. The device of claim 1, wherein a predetermined constantinter-spacer distance that is larger than the size of the analyte by afactor that is at least 2 times, at least 6 times, at least 8 times, atleast 10 times, at least 20 times, at least 40 times, at least 60 times,at least 80 times, or at least 100 times.
 35. The device of claim 1,wherein the height of light-guiding spacer is 1 um, 2 um, 5 um, 10 um,30 um, 50 um, 100 um, 200 um, 500 um, 1,000 um, 2,000 um, 5,000 um,10,000 um, or in a range between any of the two values.
 36. The deviceof claim 1, wherein the spacers are arranged in periodic array with aperiod of 1 um, 2 um, 5 um, 10 um, 30 um, 50 um, 100 um, 200 um, 500 um,1,000 um, 2,000 um, 5,000 um, 10,000 um, or in a range between any ofthe two values.
 37. The device of claim 1, wherein the LGS has a pillarshape with its ends substantially flat.
 38. The device of claim 1,wherein one or both of the ends of the LGS are fixed with one or both ofthe plates by bonding, fusing, made from a single piece, or othermethods that connect LGS to the plates.
 39. The device of claim 1,wherein the shape of the lateral cross-section of LGS includes, notlimited to circular, rectangle, square, triangle, polygon, alphabets,numbers, or a combination of thereof.
 40. The device of claim 2, whereinthe average lateral cross-section of each light-guiding spacer (LGS) is1 um{circumflex over ( )}2 (micron-square), 10 um{circumflex over ( )}2,20 um{circumflex over ( )}2, 30 um{circumflex over ( )}2, 50um{circumflex over ( )}2, 100 um{circumflex over ( )}2, 150um{circumflex over ( )}2, 200 um{circumflex over ( )}2, 300um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, 100,000 um{circumflex over ( )}2, 200,000um{circumflex over ( )}2, 500,000 um{circumflex over ( )}2, 1um{circumflex over ( )}2, 2 um{circumflex over ( )}2, 5 um{circumflexover ( )}2, 10 um{circumflex over ( )}2, 50 um{circumflex over ( )}2, orin a range between any of the two values.
 41. The device of claim 2,wherein the average lateral cross-section of each light-guiding spaceris 1 um{circumflex over ( )}2 (micron-square), 10 um{circumflex over( )}2, 20 um{circumflex over ( )}2, 30 um{circumflex over ( )}2, 50um{circumflex over ( )}2, 100 um{circumflex over ( )}2, 150um{circumflex over ( )}2, 200 um{circumflex over ( )}2, 300um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, 100,000 um{circumflex over ( )}2, 200,000um{circumflex over ( )}2, or in a range between any of the two values.42. The device of claim 2, wherein the average lateral cross-section ofeach light-guiding spacer is 1 um{circumflex over ( )}2 (micron-square),10 um{circumflex over ( )}2, 20 um{circumflex over ( )}2, 30um{circumflex over ( )}2, 50 um{circumflex over ( )}2, 100 um{circumflexover ( )}2, 150 um{circumflex over ( )}2, 200 um{circumflex over ( )}2,300 um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, 10,000 um{circumflex over ( )}2, 30,000um{circumflex over ( )}2, or in a range between any of the two values.43. The device of claim 2, wherein the average lateral cross-section ofeach light-guiding spacer is 1 um{circumflex over ( )}2 (micron-square),10 um{circumflex over ( )}2, 20 um{circumflex over ( )}2, 30um{circumflex over ( )}2, 50 um{circumflex over ( )}2, 100 um{circumflexover ( )}2, 150 um{circumflex over ( )}2, 200 um{circumflex over ( )}2,300 um{circumflex over ( )}2, 500 um{circumflex over ( )}2, 1000um{circumflex over ( )}2, 2000 um{circumflex over ( )}2, 5000um{circumflex over ( )}2, or in a range between any of the two values.44. The device of claim 1, wherein the average lateral cross-section ofeach light-guiding spacer is larger than the wavelength of the lightthat goes through the reference region, by 1 fold, 2 fold, 3 fold, 5fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000fold, 5000 fold or in a range between any of the two values.
 45. Thedevice of claim 1, wherein the average lateral cross-section of eachlight-guiding spacer is larger than the wavelength of the light thatgoes through the reference region, by 1 fold, 2 fold, 3 fold, 5 fold, 10fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, or in a rangebetween any of the two values.
 46. The device of claim 1, wherein thereference region is less than the size of the minimum lateralcross-section of the light guiding pillar. One advantage is to avoid orreduce light scattering the light guiding sidewall to affect thereference signal.
 47. The device of claim 1, wherein the minimumdistance between the edge of the light guiding spacer and that of thereference region is 1 um (micron), 2 um, 3 um, 5 um, 10 um, 20 um, 30um, 50 um, 100 um, 200 um, 500 um, 1000 um, or in a range between any ofthe two values.
 48. The device of claim 1, wherein the minimum distancebetween the edge of the light guiding spacer and that of the referenceregion is 1 um (micron), 2 um, 3 um, 5 um, 10 um, 20 um, 30 um, 50 um,100 um, 200 um, or in a range between any of the two values.
 49. Thedevice of claim 1, wherein the minimum distance between the edge of thelight guiding spacer and that of the reference region is 1 um (micron),2 um, 3 um, 5 um, 10 um, 20 um, 30 um, 50 um, or in a range between anyof the two values.
 50. The device of claim 2, wherein the minimumdistance between the edge of the light guiding spacer and that of thereference region is larger than the wavelength, that goes through thereference region, by 1 fold, 2 fold, 3 fold, 5 fold, 10 fold, 20 fold,50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000 fold or in arange between any of the two values.
 51. The device of claim 3, whereinthe minimum distance between the edge of the light guiding spacer andthat of the reference region is larger than the wavelength, that goesthrough the sampling region, by 1 fold, 2 fold, 3 fold, 5 fold, 10 fold,20 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000 fold orin a range between any of the two values.
 52. The device of claim 1,wherein the ratio of the reference region area and the light guidingspacer area is 3/10, 2/5, 1/2, 3/5, 7/10, 4/5, or in a range between anyof the two values.
 53. The device of claim 1, wherein the edge of thesampling region is a distance away from the edge of the light guidingpillar.
 54. The device of claim 1, wherein the area of the samplingregion is 3/5, 7/10, 4/5, 9/10, 1, 11/10, 6/5, 13/10, 7/5, 3/2, or inthe range between any of the two values, of the periodic inter spacerdistance.
 55. The device of claim 1, wherein the distance between theedge of the sampling region and that of the light guiding spacer is 1/5,3/10, 2/5, 1/2, 3/5, 7/10, 4/5, 9/10, 1, or in the rage between any ofthe two values, of the light guiding spacer area.
 56. The device ofclaim 1, wherein the distance between the edge of the sampling regionand that of the light guiding spacer is larger than the wavelength, thatgoes through the reference region, by 1 fold, 2 fold, 3 fold, 5 fold, 10fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000fold or in a range between any of the two values.
 57. The device ofclaim 2, wherein the distance between the edge of the sampling regionand that of the light guiding spacer is larger than the wavelength, thatgoes through the sampling region, by 1 fold, 2 fold, 3 fold, 5 fold, 10fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000fold or in a range between any of the two values.
 58. The device ofclaim 1, wherein the distance between the edge of sampling area and thereference region is 1 um (micron), 2 um, 3 um, 5 um, 10 um, 20 um, 30um, 40 um, 50 um, 100 um, 200 um, 500 um, 1000 um or in the rangebetween any of the two values.
 59. The device of claim 1, wherein thedistance between the edge of sampling area and the reference region isfrom 30 um (micron) to 50 um, 20 um to 60 um, 10 um to 70 um, 5 um to 75um, or in the range between any of the two values.
 60. The device ofclaim 1, wherein the distance between the edge of sampling area and thereference region is larger than the wavelength, that goes through thereference region, by 1 fold, 2 fold, 3 fold, 5 fold, 10 fold, 20 fold,50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 5000 fold or in arange between any of the two values.
 61. The device of claim 1, whereinthe distance between the edge of sampling area and the reference regionis 2/5, 1/2, 3/5, 7/10, 4/5, 9/10, 1, 11/10, 6/5, 13/10, 7/5, 3/2, 8/5,17/10, or in the range between any of the two values, of the lightguiding spacer area.
 62. The device of claim 2, wherein the distancebetween the edge of sampling area and the reference region is largerthan the wavelength of the light that goes through the reference region,by 1 fold, 2 fold, 3 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold,200 fold, 500 fold, 1000 fold, 5000 fold or in a range between any ofthe two values.
 63. The device of claim 3, wherein the distance betweenthe edge of sampling area and the reference region is larger than thewavelength, that goes through the sampling region, by 1 fold, 2 fold, 3fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, 200 fold, 500 fold,64. The device of claim 1, wherein the analyte is a biomarker, anenvironmental marker, or a foodstuff marker.
 65. The device of claim 1,wherein the analyte is a biomarker indicative of the presence orseverity of a disease or condition.
 66. The device of claim 1, whereinthe analyte is a cell, a protein, or a nucleic acid.
 67. The device ofclaim 2, wherein the analyte is hemoglobin.
 68. The device of claim 1,wherein the analyte comprises proteins, peptides, nucleic acids,synthetic compounds, inorganic compounds, organic compounds, bacteria,virus, cells, tissues, nanoparticles, and other molecules, compounds,mixtures and substances thereof.
 69. The device of claim 1, wherein thesample is original, diluted, or processed forms of: bodily fluids,stool, amniotic fluid, aqueous humour, vitreous humour, blood, wholeblood, fractionated blood, plasma, serum, breast milk, cerebrospinalfluid, cerumen, chyle, chime, endolymph, perilymph, feces, gastric acid,gastric juice, lymph, mucus, nasal drainage, phlegm, pericardial fluid,peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen,sputum, sweat, synovial fluid, tears, vomit, urine, or exhaled breathcondensate.
 70. The device of claim 1, wherein the sample is original,diluted, or processed forms of blood.
 71. The device of claim 1, whereinthe sample comprises whole blood.
 72. The device of claim 1, wherein theinter spacer distance (SD) is equal or less than about 150 um(micrometer).
 73. The device of claim 1, wherein the inter spacerdistance (SD) is equal or less than about 100 um (micrometer).
 74. Thedevice of claim 1, wherein the fourth power of the inter-spacer-distance(ISD) divided by the thickness (h) and the Young's modulus (E) of theflexible plate (ISD⁴/(hE)) is 5×10⁶ um³/GPa or less.
 75. The device ofclaim 1, wherein the fourth power of the inter-spacer-distance (ISD)divided by the thickness (h) and the Young's modulus (E) of the flexibleplate (ISD⁴/(hE)) is 5×10⁵ um³/GPa or less.
 76. The device of claim 1,wherein the spacers have pillar shape, a substantially flat top surface,a predetermined substantially uniform height, and a predeterminedconstant inter-spacer distance that is at least about 2 times largerthan the size of the analyte, wherein the Young's modulus of the spacerstimes the filling factor of the spacers is equal or larger than 2 MPa,wherein the filling factor is the ratio of the spacer contact area tothe total plate area, and wherein, for each spacer, the ratio of thelateral dimension of the spacer to its height is at least 1 (one). 77.The device of claim 1, wherein the spacers have pillar shape, asubstantially flat top surface, a predetermined substantially uniformheight, and a predetermined constant inter-spacer distance that is atleast about 2 times larger than the size of the analyte, wherein theYoung's modulus of the spacers times the filling factor of the spacersis equal or larger than 2 MPa, wherein the filling factor is the ratioof the spacer contact area to the total plate area, and wherein, foreach spacer, the ratio of the lateral dimension of the spacer to itsheight is at least 1 (one), wherein the fourth power of theinter-spacer-distance (ISD) divided by the thickness (h) and the Young'smodulus (E) of the flexible plate (ISD⁴/(hE)) is 5×10⁶ um³/GPa or less.78. The device of device claim 1, wherein the ratio of the inter-spacingdistance of the spacers to the average width of the spacer is 2 orlarger, and the filling factor of the spacers multiplied by the Young'smodulus of the spacers is 2 MPa or larger.
 79. The device of claim 1,wherein one or both plates comprises a location marker, either on asurface of or inside the plate, that provide information of a locationof the plate.
 80. The device of claim 1, wherein one or both platescomprises a scale marker, either on a surface of or inside the plate,that provide information of a lateral dimension of a structure of thesample and/or the plate.
 81. The device of claim 1, wherein one or bothplates comprises an image marker, either on a surface of or inside theplate that assists an imaging of the sample.
 82. The device of claim 2,wherein the sample is original, diluted, or processed forms of: bodilyfluids, stool, amniotic fluid, aqueous humour, vitreous humour, blood,whole blood, fractionated blood, plasma, serum, breast milk,cerebrospinal fluid, cerumen, chyle, chime, endolymph, perilymph, feces,gastric acid, gastric juice, lymph, mucus, nasal drainage, phlegm,pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva,sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, orexhaled breath condensate.
 83. The device of claim 2, wherein the sampleis original, diluted, or processed forms of blood.
 84. The device ofclaim 2, wherein the sample comprises whole blood.
 85. The device ofclaim 1, wherein the sample is a biological sample, a chemical sample,an environmental sample, or a foodstuff sample.